Ligands selective to alpha 6 subunit-containing gabaa receptors and their methods of use

ABSTRACT

Provided herein are novel pyrazolo-quinolinone compounds and method of using such compounds to treat disorders such as neuropsychiatric disorders with sensorimotor gating deficits, such as schizophrenia, tic disorders, attention deficit hyperactivity disorder, obsessive compulsive disorder, panic disorder, Huntington&#39;s disease and nocturnal enuresis;depression; temporomandibular myofascial pain; disorders of trigeminal nerve, such as trigeminal neuralgia and trigeminal neuropathy; migraine; and tinnitus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNos. 62/170,552, filed on Jun. 3, 2015, and 62/307,836, filed on Mar.14, 2016, the entire contents of which are hereby incorporated byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support under NIHgrant number 1 R01 MH09463-01A1. The United States government hascertain rights to this invention.

This invention was made with Taiwan government support from the Ministerof Science and Technology under grant number MOST 104-2923-B002-006-MY3and MOST 105-2325-6002-004.

BACKGROUND

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitterin the central nervous system. GABA is released from GABAergic synapsesand mediates its effect by interacting with GABA receptors. GABAreceptors can be divided into two main classes: (1) GABA_(A) receptors,which are members of the ligand-gated ion channel superfamily; and (2)GABA_(B) receptors, which are members of the G-protein linked receptorsuperfamily.

GABA_(A) receptors are composed of five subunits that form a centralchloride channel. Binding of GABA to these receptors opens the chloridechannel and usually causes an influx of chloride ions into the neuronsand thus, an inhibition of their electrical activity. GABA_(A)receptors, thus, predominantly function to inhibit and regulate neuronalactivity, and fulfill other roles in non-neuronal cells. In themammalian genome a total of 19 subunits (6α, 3β, 3γ, δ, ε, π, θ, 3ρ)belonging to 8 different subunit classes have been identified. Themajority of the receptors are composed of two α, two β, and one γsubunit. Receptors composed of two α6, two β3, and one γ2 subunit arenamed α6β3γ2 receptors. GABA_(A) receptors can also be composed of up tofive different subunits. In this case, all subunits present in thereceptor have to be mentioned to define the receptor subtype. Dependingon the regional, cellular, and subcellular distribution of theindividual subunits in the brain, a large variety of GABA_(A) receptorsubtypes with distinct subunit composition and unique pharmacology canbe formed. Due to their specific localization in the nervous system eachreceptor subtype has a specific function and can more or less stronglyinfluence the neuronal circuits on which the receptors are located andthus, modulate behavior elicited by these neuronal circuits (Olsen andSieghart, Pharmacol Rev. 2008).

GABA_(A) receptors are the site of action of a variety ofpharmacologically and clinically important drugs, such as thebenzodiazepines, barbiturates, neuroactive steroids, anesthetics, andalso some convulsants. These drugs interact with a multitude ofallosteric binding sites at GABA_(A) receptors, many of which so farhave not been identified, and by that, enhance or reduce GABA-inducedchloride flux. For benzodiazepines, the binding sites are known to be atα+/γ− extracellular interfaces of α1,2,3,5, and γ2 containing receptors.The modulatory binding sites for pyrazoloquinolinones are at theextracellular α+/β− interfaces of all GABA_(A) receptor subtypes thatcontain such interfaces (see FIG. 1).

Whereas benzodiazepines and pyrazoloquinolinones can only allostericallymodulate GABA-induced chloride flux, other compounds such asbarbiturates, neuroactive steroids and anesthetics exhibit a biphasiceffect. At low concentrations, they allosterically modulate GABA-inducedchloride currents, at higher concentrations they also can directly openthe GABA_(A) receptor-associated chloride channel in the absence ofGABA. These latter compounds are thus much more toxic than thebenzodiazepines or pyrazoloquinolinones that only can modulate ongoingGABAergic activity.

SUMMARY

In an aspect the invention provides a compound according to Formula I:

wherein

-   each X is independently C or N;-   R′₂, R′₃ and R′₄ are independently selected from H, C₁₋₄ alkyl, C₁₋₄    alkoxy, halogen, NR₁₀R₁₁,-   NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁;-   R₆, R₇, R₈ and R₉ are independently selected from H, C₁₋₄ alkyl,    C₁₋₄ alkoxy, halogen,-   NR₁₀R₁₁, NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁, or R₆ and R₇ or R₇ and R₈ can form a 4-6 member ring;-   R₁₀ and R₁₁ are independently selected from H and C₁₋₄ alkyl; and-   R_(N) is H or C₁₋₄ alkyl;

In an aspect the present invention provides a compound according toFormula (II):

wherein

-   each X is independently C or N;-   R′₂, R′₃ and R′₄ are independently selected from H, C₁₋₄ alkyl, C₁₋₄    alkoxy, halogen, NR₁₀R₁₁,-   NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁;-   R₆, R₇, R₈ and R₉ are independently selected from H, C₁₋₄ alkyl,    C₁₋₄ alkoxy, and halogen;-   NR₁₀R₁₁, NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁; or R₆ and R₇ or R₇ and R₈ can form a 4-6 member ring;-   R₁₀ and R₁₁ are independently selected from H and C₁₋₄ alkyl; and-   R_(N) is H or C₁₋₄ alkyl;-   wherein at least one of R′₂, R′₃, R′₄, R₆, R₇, R₈ , R₉, R₁₀, R₁₁ and    R_(N) contains at least one deuterium.

In an aspect the invention provides a pharmaceutical compositioncomprising the compounds described herein and a pharmaceuticallyacceptable carrier.

In an aspect the invention provides a method of treating diseases and/orconditions which are regulated by the α6-GABA_(A) receptor comprisingadministering a therapeutically effective amount of a compound describedherein to a subject in need thereof.

In an aspect the invention provides a method of treating a diseaseand/or condition comprising administering a therapeutically effectiveamount of a compound described herein to a subject in need thereof;wherein the disease is modulated by the α6-subunit GABA_(A) receptor.

Other aspects of the invention will become apparent by consideration ofthe detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The scheme represents an extracellular domain of a receptor withthe most common two α, two β, and one γ composition and arrangement.Plus and minus signs indicate the subunits' principal, or plus- andcomplementary, or minus-sites. The two binding sites labelled with(GABA) are the GABA binding sites, (Bz) denotes the high affinitybenzodiazepine binding site, and (mPQ) is the “modulatoryprazoloquinolinone” binding site at the α+/β− interface.

FIG. 2. Dose response curves of the change of GABA EC3 currents(modulation, referenced to 100% for the EC3 current) by increasingcompound concentrations in the indicated receptor subtypes. A, C, E:Compound 6, LAU 463, and LAU 159 preferentially modulate GABA currentsof α6β3γ2 receptors compared to the corresponding α1, 2, 3, 4, 5β3γ2receptors. B, D, F: Measurements in the α1,4,6β3δ receptors (α1,4,6 arethe major subunits that co-assemble with the delta subunit) indicatethat compound 6 and LAU 159 also preferentially modulate the α6β3δreceptor, compared to those containing α1 or α4 subunits. LAU 463 exertssimilar effects on all three tested delta-containing receptors that aremuch smaller than those in the α6β3δ receptor.

FIG. 3. A: Dose response curve of the modulation of GABA EC3 (100%)currents by increasing DK-I-86-1 compound concentrations in the α6β3γ2receptor. Other subtypes were investigated only at 1 and 10 μM to testselectivity. DK-I-86-1 preferentially modulates α6β3γ2 receptors. B:Receptor potentiation in the α6β3δ receptor by DK-I-86-1, note that dueto low currents of these receptors it was measured at EC20 and thusappears smaller, however, data indicate that this receptor pool also ismodulated.

FIG. 4. Effects of Compound 6, LAU 159, LAU 463 and Comopound 11,examples of positive allosteric modulators (PAMs) selective to α6GABARs,on the impairment of prepulse inhibition of the startle reflex (PPI)induced by methamphetamine (METH). The magnitudes of PPI in the startleresponse to a 115 dB acoustic stimulation paired with a prepulse of 71dB (71-115 dB) or 77 dB (77-115 dB) ahead in 120 ms were measured asdescribe in Materials and Methods. Mice were pretreated with the testedcompound 10 mg/kg (i. p.) or vehicle for 15 min followed bymethamphetamine (METH, 2 mg/kg, i. p.) for 10 min. Tested compounds weredissolved in a vehicle containing 20% DMSO, 20% Cremophor® EL(polyoxyethylene castor, Sigma-Aldrich) and 60% normal saline.***p<0.001 vs. the Vehicle without METH group; ^(#)p<0.05,^(###)p<0.001, vs. the Vehicle with METH group with the 71-115 dB or77-115 dB protocol (Student's t test). N=6.

FIG. 5 is a graph showing that Compounds 6 (C6) and Compound 11 (C11)when given by i.p. injection at 10 mg/kg significantly rescuedMETM-impaired PPI. Effects of both compounds were prevented byintra-cerebellar microinjection of furosemde (10 nmol), a α6GABA_(A)Rantagonist. The measurement and analyses of PPI impairment are the sameas in FIG. 4. ***p<0.001 vs. the Vehicle without METH group;^(###)p<0.001, vs. the Vehicle with METH group; ^(&&)p<0.01,^(&&&)p<0.001 vs. the C6 with METH group.

FIG. 6. Effects of 1-29 and DK-I-56-1 (—OCD3 derivatives of Compound 6),DK-I-59-1 (a —OCD3 derivative of of LAU 159) and DK-158-1 (a —OCD3derivative of of LAU 159) on METH-impaired PPI. The measurement andanalyses of PPI impairment are the same as in FIG. 4. ***p<0.001 vs. theVehicle without METH group; ^(#)p<0.05; ^(##)p<0.01, ^(###)p<0.001, vs.the Vehicle with METH group.

FIG. 7. Effects of Compound 6 on total IR beam breaks in mice undertreatment with saline or methamphetamine (2 mg/kg, i.p.) for 60 min.Mice were pretreated with Compound 6 (C6, 3 mg/kg, i.p.) or vehicle (V),or Compound 6+ furosemide (10 nmol, intra-cerebellar injection) for 10min before METH/saline injection. The locomotor activity was measured bythe number of infra-red (IR) beam interruptions in the open fieldchamber every 5 min. The ordinate is total IR beam interruptionsrecorded during the 60 min-METH treatment period. The number of animaltestd in each group is denoted above each bar. **p<0.01 vs. the Vehiclewith METH group (Student's t test).

FIG. 8. Total scores of stereotypy behaviors after injection ofapomorphine (1 mg/kg, s.c.) or normal saline in groups pretreated withCompound 6 (C6, 3 mg/kg, i.p.) or vehicle (V). Stereotypy climbingbehaviors were scored as described in Materials and Methods every 5 minfor 1 min before and after apomorphine injection. The ordinate is totalscores recorded during the 45 min-apomorphine treatmetn perioid indifferent groups. ***p<0.001 vs. the Vehicle without apomorphine group(Student's t test).

FIG. 9. Effect of Compound 6 on the grip strength (muscle power) ofmice. The grip strength of forepaws of the mouse was measured by a gripstrength meter three times every 2 min and averaged. The grip strengthof the mouse was measaured before (pre) or 15 min after treatment withCompound 6 (C6, 3 mg/kg) and vehicle (V).

FIG. 10. Effect of Compound 6 on the performance of motor coordinationin mice. Motor coordination was measured by the latency to fall in therotarod test before before (pre) or 15 min after treatment with Compound6 (C6, 3 mg/kg) and vehicle (V). The mouse was pre-trained until thelatency to fall was greater than 120 sec.

FIG. 11. Effect of Compound 6 on the performance of mice in a sedationassessment. The sedative effect was assessed by the latency for a mouseto step off from a 3 cm-high platform in a 60 sec-session with theformula: Sedation effect %=(Test latency−baseline latency)/(60−baselinelatency)×100%. Compound 6 (C6, 3 mg/kg, i.p.) or vehicle (V) was given10 min before the tests.

FIG. 12. Effects of Compound 6 on the performance of mice in theelevated plus maze (EPM) test, a measurement for its possible anxiolyticactivity. It was assessed by the ratio of the duration (a) or entrydistance (b) in open arms of the EPM apparatus. The rearing number ofmice (c) in the EPM apparatus was also measured to evaluate their motorfunction. Compound 6 (3 mg/kg or 10 mg/kg i.p.) was given 10 min beforethe tests.

FIG. 13. Compound 6 reduces the number of capsaicin-increased c-fos itcells in the trigeminal nucleus caudalis. Rats were given i.p. injectionof 3 or 10 mg/kg of Compound 6 or its vehicle. The capsaicin (10 nmol,100 μL) was injected by intracisterna injection. Two hours aftercapsicine injection, the total number of c-Fos-ir TCC neurons in threegroups were estimated based on the formular derived in our previousstudy (Fan et al., 2012): 16(N1+N2)/2+53(N2+N3)/2, where N1, N2, and N3were the c-Fos-ir neuronal numbers measured at the level of 0.6, −1.2,and −9 mm from the obex, respectively. Data are mean mean+SE. (One wayANOVA with Tukey posthoc test), n=2 in the vehicle group and n=3 in 10and 30 mg/kg treated groups, respectively.

FIG. 14A shows the prophylactic antinociceptive effect of subchronictreatment with DK-I-56-1 in rats with unilateral chronic constrictioninjury to the infraorbital nerve (IoN-CCl), as assessed by von Freyfilaments. FIG. 14B represents the significances between the treatment(IoN-CCl-DK-I-56-1) and control group (IoN-CCl-Placebo) of operatedanimals. Each point is the mean±S.E.M. The number of analyzed animalsfor Sham-Placebo, Sham-DK-I-56-1, IoN-CCl-Placebo and Ion-CCl-DK-I-56-1group was 9, 11, 11, and 12, respectively. *P<0.05 and **P<0.01 comparedto IoN-CCl-Placebo group; two-way ANOVA on the data for the given day,followed by Student Newman Keuls post-hoc test. Further details aregiven in Example 10.

FIG. 15 shows the total time the rats spent in face grooming during bodygrooming during 10 min of recording. Further details are given inExample 10.

FIG. 16 are graphs (A)-(E) showing concentration-time curves for theplasma and brain levels, as well as the calculated pharmacokineticparameters in plasma and brain for various compounds.

FIG. 17 is a graph of viability versus log[concentration] for cytotoxicanalysis for various compounds.

FIG. 18 is a graph of viability versus log[concentration] for cytotoxicanalysis for various compounds.

FIG. 19 is a graph of viability versus log[concentration] for cytotoxicanalysis for various compounds.

FIG. 20 is a graph of the time mice performed in the rotorod assay.

FIG. 21 is a graph of the half-life of compounds in human livermicrosomes and mouse liver microsomes.

FIG. 22 is a graph of the metabolic rate of compounds of human livermicrosomes and mouse liver microsomes.

FIG. 23 shows compounds according to the present invention.

FIG. 24. Efficacy (% of modulation of control GABA EC3 current=100%) oftest compounds at different recombinant rat αxβx, αxβ3γ2 and αβδreceptors expressed in Xenopus laevis oocytes. Values are reported asmean±SEM.

FIGS. 25A and 25B show synthetic schemes for the preparation ofcompounds.

DETAILED DESCRIPTION

In embodiments this disclosure provides novel pyrazoloquinolinonecompounds. In embodiments this disclosure provides pyrazoloquinolinonecompounds with selective modulatory activity at GABA_(A) α6β3γ2receptors. In embodiments, this invention provides pyrazoloquinolinonecompounds with selective modulatory activity at GABA_(A) α6β1γ2receptors. In embodiment, this invention provides pyrazoloquinolinonecompounds with selective modulatory activity at GABA_(A) α6βδ receptors.In embodiments the invention provides pyrazoloquinolinone compounds withselective modulatory activity at more than one GABA_(A) receptorselected from α6β3γ2, α6β1γ2, and α6βδ.

In embodiments this disclosure describes a method of treating a disorderwith a pyrazoloquinolinone compound including, but not limited to,neuropsychiatric disorders with sensorimotor gating deficits, such asschizophrenia, tic disorders, attention deficit hyperactivity disorder,obsessive compulsive disorder, panic disorder, Huntington's disease andnocturnal enuresis; depression; temporomandibular myofascial pain;disorders of trigeminal nerve, such as trigeminal neuralgia andtrigeminal neuropathy; migraine; and tinnitus.

In embodiments this disclosure describes a method of treatingneuropsychiatric disorders with sensorimotor gating deficits, such asschizophrenia, tic disorders, attention deficit hyperactivity disorder,obsessive compulsive disorder, panic disorder, Huntington's disease andnocturnal enuresis; depression; temporomandibular myofascial pain;disorders of trigeminal nerve, such as trigeminal neuralgia andtrigeminal neuropathy; migraine; and tinnituswith a compound with aselective positive allosteric modulation at α6 subunit-containingGABA_(A) receptors.

In embodiments the present invention provides compounds with thefollowing structure:

wherein

-   each X is independently C or N;-   R′₂, R′₃ and R′₄ are independently selected from H, C₁₋₄ alkyl, C₁₋₄    alkoxy, halogen, NR₁₀R₁₁,-   NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁;-   R₆, R₇, R₈ and R₉ are independently selected from H, C₁₋₄ alkyl,    C₁₋₄ alkoxy, halogen,-   NR₁₀R₁₁, NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁, or R₆ and R₇ or R₇ and R₈ can form a 4-6 member ring;-   R₁₀ and R₁₁ are independently selected from H and C₁₋₄ alkyl; and-   R_(N) is H or C₁₋₄ alkyl.

In embodiments, the present invention also provides compounds of Formula(II):

wherein

-   each X is independently C or N;-   R′₂, R′₃ and R′₄ are independently selected from H, C₁₋₄ alkyl, C₁₋₄    alkoxy, halogen, NR₁₀R₁₁,-   NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁;-   R₆, R₇, R₈ and R₉ are independently selected from H, C₁₋₄ alkyl,    C₁₋₄ alkoxy, halogen,-   NR₁₀R₁₁, NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁, or R₆ and R₇ or R₇ and R₈ can form a 4-6 member ring;-   R₁₀ and R₁₁ are independently selected from H and C₁₋₄ alkyl; and-   R_(N) is H or C₁₋₄ alkyl;-   wherein at least one of R′₂, R′₃, R′₄, R₆, R₇, R₈ , R₉, R₁₀, R₁₁ and    R_(N) contains at least one deuterium.

In embodiments these compounds rescue METH-induced PPI impairment inmice, a mouse model mimicking sensorimotor gating deficit in severalneuropsychiatric disorders, such as, but not limited to,neuropsychiatric disorders with sensorimotor gating deficits, such asschizophrenia, tic disorders, attention deficit hyperactivity disorder,obsessive compulsive disorder, panic disorder, Huntington's disease, andnocturnal enuresis.

In embodiments these compounds also reduce the number of activatedneurons in trigeminal nucleus caudalis (TNC) induced by intra-cisternalcapsaicin injection, an animal model mimicking migraine, and rats withreduced α6 GABA_(A)Rs in trigeminal ganglia were hypersensitive to TMJinflammation), as well as in animal model of trigeminal neuropathicpain.

In embodiments these compounds modulate hearing disorders, such as butnot limited to tinnitus, as α6-containing GABA_(A) receptors are alsoexpressed in the cochlear nucleus.

Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock,Comprehensive Organic Transformations, VCH Publishers, Inc., New York,1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition,Cambridge University Press, Cambridge, 1987; the entire contents of eachof which are incorporated herein by reference.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,heterocyclylcarbonyl, arylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted (e.g., with one or moresubstituents).

The term “alkyl” refers to a straight or branched hydrocarbon chain,containing the indicated number of carbon atoms. For example, C₁-C₁₂alkyl indicates that the alkyl group may have from 1 to 12 (inclusive)carbon atoms, and C₁-C₄ alkyl indicates that the alkyl group may havefrom 1 to 4 (inclusive) carbon atoms. An alkyl group may be optionallysubstituted. Examples of C₁-C₄ alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, and tert-butyl.

The term “alkenyl” refers to a straight or branched hydrocarbon chainhaving one or more double bonds. Examples of alkenyl groups include, butare not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl, and 3-octenylgroups. One of the double bond carbons may optionally be the point ofattachment of the alkenyl substituent. An alkenyl group may beoptionally substituted.

The term “alkynyl” refers to a straight or branched hydrocarbon chainhaving one or more triple bonds. Examples of alkynyl groups include, butare not limited to, ethynyl, propargyl, and 3-hexynyl. One of the triplebond carbons may optionally be the point of attachment of the alkynylsubstituent. An alkynyl group may be optionally substituted.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom capable of substitutioncan be substituted (e.g., with one or more substituents). Examples ofaryl moieties include, but are not limited to, phenyl, naphthyl, andanthracenyl.

The term “arylalkyl” refers to an alkyl moiety in which an alkylhydrogen atom is replaced with an aryl group. Arylalkyl includes groupsin which more than one hydrogen atom has been replaced with an arylgroup. Examples of arylalkyl groups include benzyl, 2-phenylethyl,3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

The term “cycloalkyl” as used herein refers to nonaromatic, saturated orpartially unsaturated cyclic, bicyclic, tricyclic, or polycyclichydrocarbon groups having 3 to 12 carbons (e.g., 3, 4, 5, 6 or 7 carbonatoms). Any ring atom can be substituted (e.g., with one or moresubstituents). Cycloalkyl groups can contain fused rings. Fused ringsare rings that share one or more common carbon atoms. Examples ofcycloalkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexadienyl,methylcyclohexyl, adamantyl, norbornyl, and norbornenyl.

The term “halo” or “halogen” as used herein refers to any radical offluorine, chlorine, bromine, or iodine.

The term “haloalkyl” as used herein refers to an alkyl in which one ormore hydrogen atoms are replaced with a halogen, and includes alkylmoieties in which all hydrogens have been replaced with halogens (e.g.,perfluoroalkyl such as CF₃).

The term “heteroaryl” as used herein refers to an aromatic 5-8 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatomsindependently selected from O, N, S, P, and Si (e.g., carbon atoms and1-3, 1-6, or 1-9 heteroatoms independently selected from O, N, S, P, andSi if monocyclic, bicyclic, or tricyclic, respectively). Any ring atomcan be substituted (e.g., with one or more substituents). Heteroarylgroups can contain fused rings, which are rings that share one or morecommon atoms. Examples of heteroaryl groups include, but are not limitedto, radicals of pyridine, pyrimidine, pyrazine, pyridazine, pyrrole,imidazole, pyrazole, oxazole, isoxazole, furan, thiazole, isothiazole,thiophene, quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline,indole, isoindole, indolizine, indazole, benzimidazole, phthalazine,pteridine, carbazole, carboline, phenanthridine, acridine,phenanthroline, phenazine, naphthyridines, and purines.

The term “heterocyclyl” as used herein refers to a nonaromatic,saturated or partially unsaturated 3-10 membered monocyclic, 8-12membered bicyclic, or 11-14 membered tricyclic ring system having 1-3heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, S, Si,and P (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, S,Si, and P if monocyclic, bicyclic, or tricyclic, respectively). Any ringatom can be substituted (e.g., with one or more substituents).Heterocyclyl groups can contain fused rings, which are rings that shareone or more common atoms. Examples of heterocyclyl groups include, butare not limited to, radicals of tetrahydrofuran, tetrahydrothiophene,tetrahydropyran, piperidine, piperazine, morpholine, pyrroline,pyrimidine, pyrrolidine, indoline, tetrahydropyridine, dihydropyran,thianthrene, pyran, benzopyran, xanthene, phenoxathiin, phenothiazine,furazan, lactones, lactams such as azetidinones and pyrrolidinones,sultams, sultones, and the like.

The term “hydroxy” refers to an —OH radical. The term “alkoxy” refers toan —O-alkyl radical. The term “aryloxy” refers to an —O-aryl radical.The term “haloalkoxy” refers to an —O-haloalkyl radical.

The term “substituent” refers to a group “substituted” on an alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, arylalkyl, orheteroaryl group at any atom of that group. Suitable substituentsinclude, without limitation: acyl, acylamido, acyloxy, alkoxy, alkyl,alkenyl, alkynyl, amido, amino, carboxy, cyano, ester, halo, hydroxy,imino, nitro, oxo (e.g., C═O), phosphonate, sulfinyl, sulfonyl,sulfonate, sulfonamino, sulfonamido, thioamido, thiol, thioxo (e.g.,C═S), and ureido. In embodiments, substituents on a group areindependently any one single, or any combination of the aforementionedsubstituents. In embodiments, a substituent may itself be substitutedwith any one of the above substituents.

The above substituents may be abbreviated herein, for example, theabbreviations Me, Et and Ph represent methyl, ethyl, and phenyl,respectively. A more comprehensive list of the abbreviations used byorganic chemists appears in the first issue of each volume of theJournal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations used by organic chemistsof ordinary skill in the art, are hereby incorporated by reference.

For compounds, groups and substituents thereof may be selected inaccordance with permitted valence of the atoms and the substituents,such that the selections and substitutions result in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they optionally encompasssubstituents resulting from writing the structure from right to left,e.g., —CH₂O— optionally also recites —OCH₂—.

In accordance with a convention used in the art, the group:

is used in structural formulas herein to depict the bond that is thepoint of attachment of the moiety or substituent to the core or backbonestructure.

In the context of treating a disorder, the term “effective amount” asused herein refers to an amount of the compound or a compositioncomprising the compound which is effective, upon single or multiple doseadministrations to a subject, in treating a cell, or curing,alleviating, relieving, or improving a symptom of the disorder in asubject. An effective amount of the compound or composition may varyaccording to the application. In the context of treating a disorder, aneffective amount may depend on factors such as the disease state, age,sex, and weight of the individual, and the ability of the compound toelicit a desired response in the individual. In an example, an effectiveamount of a compound is an amount that produces a statisticallysignificant change in a given parameter as compared to a control, suchas in cells (e.g., a culture of cells) or a subject not treated with thecompound.

It is specifically understood that any numerical value recited herein(e.g., ranges) includes all values from the lower value to the uppervalue, i.e., all possible combinations of numerical values between thelowest value and the highest value enumerated are to be considered to beexpressly stated in this application. For example, if a concentrationrange is stated as 1% to 50%, it is intended that values such as 2% to40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in in thisspecification. These are only examples of what is specifically intended.

Compounds

Some pyrazoloquinolinone compounds have selective activity at GABA_(A)α6β3γ2 receptors. In addition, some also modulate α6βδ receptors. In anaspect, the present invention relates to compounds that are selectivefor such GABA_(A) α6-containing receptors.

In embodiments, the present invention provides compounds of Formula (I):

wherein

-   each X is independently C or N;-   R′₂, R′₃ and R′₄ are independently selected from H, C₁₋₄ alkyl, C₁₋₄    alkoxy, halogen, NR₁₀R₁₁,-   NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁;-   R₆, R₇, R₈ and R₉ are independently selected from H, C₁₋₄ alkyl,    C₁₋₄ alkoxy, halogen,-   NR₁₀R₁₁, NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁, or R₆ and R₇ or R₇ and R₈ can form a 4-6 member ring;-   R₁₀ and R₁₁ are independently selected from H and C₁₋₄ alkyl; and-   R_(N) is H or C₁₋₄ alkyl.

In embodiments, the compound of Formula (I) is not

The present invention also provides compounds of Formula (II):

wherein

-   each X is independently C or N;-   R′₂, R′₃ and R′₄ are independently selected from H, C₁₋₄ alkyl, C₁₋₄    alkoxy, halogen, NR₁₀R₁₁,-   NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁;-   R₆, R₇, R₈ and R₉ are independently selected from H, C₁₋₄ alkyl,    C₁₋₄ alkoxy, halogen,-   NR₁₀R₁₁, NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or    —C(O)NR₁₀R₁₁, or R₆ and R₇ or R₇ and R₈ can form a 4-6 member ring;-   R₁₀ and R₁₁ are independently selected from H and C₁₋₄ alkyl; and-   R_(N) is H or C₁₋₄ alkyl;-   wherein at least one of R′₂, R′₃, R′₄, R₆, R₇, R₈ , R₉, R₁₀, R₁₁,    and R_(N) contains at least one deuterium.

In some embodiments, at least one of R′₂, R′₃, R′₄, R₆, R₇, R₈ , R₉,R₁₀, R₁₁, and R_(N) is a haloalkyl such as CF₃. In embodiments, at leastone of R′₂, R′₃, R′₄, R₆, R₇, R₈ , R₉, R₁₀, R₁₁, and R_(N) is C₁₋₄alkoxy, such as methoxy or —OCD₃. In embodiments, at least one of R′₂,R′₃, R′₄, R₆, R₇, R₈, and R₉ is a halogen, such as chlorine or bromine.In embodiments, R_(N) is H. In embodiments, each X is C. In embodiments,at least one X is N.

In an embodiment, the compound of Formula (I) may be:

In an embodiment, the compound of Formula (II) may be:

In an embodiment, the compound of Formulae (I) or (II) may be:

wherein R₁ and R₂ are independently H, OCH₃, OCD₃, OEt, OCF₃, F, Cl, BR,or NO₂.

In an embodiment, the compound of Formula (II) may be:

In some aspects the compounds of the present invention selectivelytarget GABA_(A) receptors of α6βγ2 composition. In some aspects, thecompounds of the present invention are allosteric modulators of theGABA_(A) receptors that are selective for the α6+/β− allostericmodulatory sites on GABA_(A) receptors.

For compounds according to the present invention, groups andsubstituents thereof may be selected in accordance with permittedvalence of the atoms and the substituents, such that the selections andsubstitutions result in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they optionally encompasssubstituents resulting from writing the structure from right to left,e.g., —CH₂O— optionally also recites —OCH₂—.

Compounds according to the present invention include compounds thatdiffer only in the presence of one or more isotopically enriched atoms.For example, compounds may have the present structures except for thereplacement of hydrogen by deuterium or tritium, or the replacement of acarbon by a ¹³C- or ¹⁴C-enriched carbon.

A compound according to the present invention can be in the form of asalt, e.g., a pharmaceutically acceptable salt. The term“pharmaceutically acceptable salt” includes salts of the activecompounds that are prepared with relatively nontoxic acids or bases,depending on the particular substituents found on the compounds.Suitable pharmaceutically acceptable salts of the compounds of thisinvention include acid addition salts which may, for example, be formedby mixing a solution of the compound according to the invention with asolution of a pharmaceutically acceptable acid such as hydrochloricacid, sulfuric acid, methanesulfonic acid, fumaric acid, maleic acid,succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid,tartaric acid, carbonic acid, or phosphoric acid. Furthermore, where thecompounds of the invention carry an acidic moiety, suitablepharmaceutically acceptable salts thereof may include alkali metalsalts, e.g. sodium or potassium salts, alkaline earth metal salts, e.g.calcium or magnesium salts; and salts formed with suitable organicligands, e.g. quaternary ammonium salts.

Neutral forms of the compounds may be regenerated by contacting the saltwith a base or acid and isolating the parent compound in a conventionalmanner. The parent form of the compound differs from the various saltforms in certain physical properties, such as solubility in polarsolvents, but otherwise the salts are equivalent to the parent form ofthe compound for the purposes of this disclosure.

In addition to salt forms, the present invention may also providecompounds according to the present invention in a prodrug form. Prodrugsof the compounds are those compounds that readily undergo chemicalchanges under physiological conditions to provide the active compounds.Prodrugs can be converted to the compounds of the present invention bychemical or biochemical methods in an ex vivo environment. For example,prodrugs can be slowly converted to the compounds of the presentinvention when placed in a transdermal patch reservoir with a suitableenzyme or chemical reagent.

Compounds according to the present invention can be, for example, anenantiomerically enriched isomer of a stereoisomer described herein.Enantiomer, as used herein, refers to either of a pair of chemicalcompounds whose molecular structures have a mirror-image relationship toeach other. For example, a compound may have an enantiomeric excess ofat least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

A preparation of a compound according to the present invention may beenriched for an isomer of the compound having a selectedstereochemistry, e.g., R or S, corresponding to a selected stereocenter.For example, the compound may have a purity corresponding to a compoundhaving a selected stereochemistry of a selected stereocenter of at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Acompound can, for example, include a preparation of a compound disclosedherein that is enriched for a structure or structures having a selectedstereochemistry, e.g., R or S, at a selected stereocenter.

In some embodiments, a preparation of a compound according to thepresent invention may be enriched for isomers (subject isomers) whichare diastereomers of the compound. Diastereomer, as used herein, refersto a stereoisomer of a compound having two or more chiral centers thatis not a mirror image of another stereoisomer of the same compound. Forexample, the compound may have a purity corresponding to a compoundhaving a selected diastereomer of at least about 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

When no specific indication is made of the configuration at a givenstereocenter in a compound, any one of the configurations or a mixtureof configurations is intended.

A compound according to the present invention can also be modified byappending appropriate functionalities to enhance selective biologicalproperties. Such modifications are known in the art and include thosethat increase biological penetration into a given biological system(e.g., blood, lymphatic system, central nervous system), increase oralavailability, increase solubility to allow administration by injection,alter metabolism, and/or alter rate of excretion. Examples of thesemodifications include, but are not limited to, esterification withpolyethylene glycols, derivatization with pivolates or fatty acidsubstituents, conversion to carbamates, hydroxylation of aromatic rings,and heteroatom substitution in aromatic rings.

Synthesis

Compounds of formulae (I) and (II) may be synthesized using commerciallyavailable starting materials according to the methods described in theexamples. Compounds according to Formula (II) may also be synthesizedaccording to the methods shown in FIG. 25A and 25B.

Other methods of synthesizing the compounds of the formulae herein willbe evident to those of ordinary skill in the art. Synthetic chemistrytransformations and protecting group methodologies (protection anddeprotection) useful in synthesizing the compounds are known in the artand include, for example, those such as described in R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof.

Evaluation of Compounds

Compounds may be analyzed using a number of methods, including receptorbinding studies, electrophysiological assays to quantify functionaleffects, and in vivo methods.

For example, the GABA_(A) subunit selectivity of compounds can beevaluated, for example, using competitive binding assays. Such assayshave been described (Choudhary et al. Mol Pharmacol. 1992, 42, 627-33;Savić et al, Progress Neuro-Psychopharmacology & Biological Psychiatry,2010, 34, 376-386). The assays involve the use of a radiolabeledcompound known to bind to GABA_(A) receptors, such as [³H]flunitrazepam.Membrane proteins can be harvested and incubated with the radiolabeledcompound, and non-specific binding can be evaluated by comparing bindingof the radiolabeled compound to another, non-labeled compound (e.g.,diazepam). Bound radioactivity can be quantified by liquid scintillationcounting. Membrane protein concentrations can be determined usingcommercially available assay kits (e.g., from Bio-Rad, Hercules,Calif.).

Compounds can also be evaluated in electrophysiological assays inXenopus oocytes. Compounds can be preapplied to the oocytes before theaddition of GABA, which can then be coapplied with the compounds until apeak response is observed. Between applications, oocytes can be washedto ensure full recovery from desensitization. For current measurements,the oocytes can be impaled with microelectrodes, and recordingsperformed using two electrode voltage clamp recordings.

The compounds may possess selective efficacy for α6βγ2 or otherα6-containing GABA_(A) receptors such that they enhance GABA elicitedcurrents to a high degree in these receptors, and to a much lesserdegree in all receptors containing α1, α2, α3, α4 and α5 subunits, asindicated in FIG. 24. The compounds may possess in addition affinity forthe benzodiazepine binding sites of all subtypes, at which they aresilent binders, meaning that they do not change GABA elicited currentsby interacting with this binding site (see FIG. 1). Other methods forevaluating compounds are known to those skilled in the art. To assess acompound's undesirable side effects (toxicity), animals may be monitoredfor overt signs of impaired neurological or muscular function. In mice,the rotorod procedure (Dunham, M. S. et al. J. Amer. Pharm. Ass. Sci.Ed. 1957, 46, 208-209) is used to disclose minimal muscular orneurological impairment. When a mouse is placed on a rod that rotates ata speed of 6 rpm, the animal can maintain its equilibrium for longperiods of time. The animal is considered intoxicated if it falls offthis rotating rod three times during a 1-min period. In rats, minimalmotor deficit is indicated by ataxia, which is manifested by anabnormal, uncoordinated gait. Rats used for evaluating toxicity areexamined before the test drug is administered, since individual animalsmay have peculiarities in gait, equilibrium, placing response, etc.,which might be attributed erroneously to the test substance. In additionto MMI, animals may exhibit a circular or zigzag gait, abnormal bodyposture and spread of the legs, tremors, hyperactivity, lack ofexploratory behavior, somnolence, stupor, catalepsy, loss of placingresponse, and changes in muscle tone.

Compositions and Routes of Administration

In another aspect, the invention provides pharmaceutical compositionscomprising one or more compounds of this invention in association with apharmaceutically acceptable carrier. Such compositions may be in unitdosage forms such as tablets, pills, capsules, powders, granules,sterile parenteral solutions or suspensions, metered aerosol or liquidsprays, drops, ampoules, auto-injector devices or suppositories; fororal, parenteral, intranasal, sublingual, or rectal administration, orfor administration by inhalation or insufflation. It is also envisionedthat compounds may be incorporated into transdermal patches designed todeliver the appropriate amount of the drug in a continuous fashion. Forpreparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture for acompound of the present invention, or a pharmaceutically acceptable saltthereof. When referring to these preformulation compositions ashomogeneous, it is meant that the active ingredient is dispersed evenlythroughout the composition so that the composition may be easilysubdivided into equally effective unit dosage forms such as tablets,pills and capsules. This solid preformulation composition is thensubdivided into unit dosage forms of the type described above containingfrom 0.1 to about 500 mg of the active ingredient of the presentinvention. Typical unit dosage forms contain from 1 to 100 mg, forexample, 1, 2, 5, 10, 25, 50, or 100 mg, of the active ingredient. Thetablets or pills of the novel composition can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an entericlayer, which serves to resist disintegration in the stomach and permitsthe inner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the compositions of the present invention maybe incorporated for administration orally or by injection includeaqueous solutions, suitably flavored syrups, aqueous or oil suspensions,and flavored emulsions with edible oils such as cottonseed oil, sesameoil, coconut oil or peanut oil, as well as elixirs and similarpharmaceutical vehicles. Suitable dispersing or suspending agents foraqueous suspensions include synthetic and natural gums such astragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinylpyrrolidone, or gelatin.

Suitable dosage level is about 0.01 to 250 mg/kg per day, about 0.05 to100 mg/kg per day, or about 0.05 to 5 mg/kg per day. The compounds maybe administered on a regimen of 1 to 4 times per day, or on a continuousbasis via, for example, the use of a transdermal patch.

Pharmaceutical compositions for enteral administration, such as nasal,buccal, rectal or, especially, oral administration, and for parenteraladministration, such as intravenous, intramuscular, subcutaneous,peridural, epidural, or intrathecal administration, are suitable. Thepharmaceutical compositions comprise from approximately 1% toapproximately 95% active ingredient, or from approximately 20% toapproximately 90% active ingredient.

For parenteral administration including intracoronary,intracerebrovascular, or peripheral vascular injection/infusionpreference is given to the use of solutions of the subunit selectiveGABA_(A) receptor agonist, and also suspensions or dispersions,especially isotonic aqueous solutions, dispersions or suspensions which,for example, can be made up shortly before use. The pharmaceuticalcompositions may be sterilized and/or may comprise excipients, forexample preservatives, stabilizers, wetting agents and/or emulsifiers,solubilizers, viscosity-increasing agents, salts for regulating osmoticpressure and/or buffers and are prepared in a manner known per se, forexample by means of conventional dissolving and lyophilizing processes.

For oral pharmaceutical preparations suitable carriers are especiallyfillers, such as sugars, for example lactose, saccharose, mannitol orsorbitol, cellulose preparations and/or calcium phosphates, and alsobinders, such as starches, cellulose derivatives and/orpolyvinylpyrrolidone, and/or, if desired, disintegrators, flowconditioners and lubricants, for example stearic acid or salts thereofand/or polyethylene glycol. Tablet cores can be provided with suitable,optionally enteric, coatings. Dyes or pigments may be added to thetablets or tablet coatings, for example for identification purposes orto indicate different doses of active ingredient. Pharmaceuticalcompositions for oral administration also include hard capsulesconsisting of gelatin, and also soft, sealed capsules consisting ofgelatin and a plasticizer, such as glycerol or sorbitol. The capsulesmay contain the active ingredient in the form of granules, or dissolvedor suspended in suitable liquid excipients, such as in oils.

Transdermal application is also considered, for example using atransdermal patch, which allows administration over an extended periodof time, e.g. from one to twenty days.

Methods of Treatment

The present invention also provides methods of treating diseases and/orconditions which are regulated by the α6GABA_(A)Rs comprisingadministering to a subject in need thereof an effective amount of acompound or composition as described herein. In embodiments, the presentinvention provides a method of treating a disease comprisingadministering to a subject in need thereof an effective amount ofcompound or composition described herein; wherein the disease isselected from the group consisting of neuropsychiatric disorders withsensorimotor gating deficits, such as schizophrenia, tic disorders,attention deficit hyperactivity disorder, obsessive compulsive disorder,panic disorder, Huntington's disease and nocturnal enuresis; depression;orofacial pains including but not limited to myofascial pain,trigeminoneuragia, and trigeminal neuropathic pain; migraine; andtinnitus.

Without wishing to be bound by theory, it is thought that the compoundsdescribed herein are functionally selective for α6β2, 3γ2 GABA_(A)receptors and are positive allosteric modulators at this subtype.(SeeFIG. 2)

Sensorimotor Gating Function

The α6subunits of GABA_(A) receptors exhibit a quite restricted regionaldistribution in the brain. α₆GABA_(A)Rs are mainly expressed incerebellar granule cells. α₆GABA_(A)Rs are located synaptically as wellas extrasynaptically and mediate inhibition induced by synaptically aswell as tonically released and spillover GABA in cerebellar cells. Theythus mediate inhibitory synaptic transmission initiated by phasicallyreleased GABA as well as providing a tonic inhibitory control of theinformation flow. The α₆GABA_(A)Rs are unresponsive to the classicalbenzodiazepine, diazepam.

PPI is a neurophysiological phenomenon in which the startle response toa stimulus (pulse) is inhibited if a weaker stimulus (prepulse) from thesame source is pre-applied within a short interval. The stimulus couldbe a sound, an airpuff, or a light. PPI is believed to be a processingprotection in a living organism, serving as a preconscious regulator ofattention, termed sensorimotor gating, to reduce the startle responsethat is harmful to the information processing. PPI can be assessed bythe motor response in either humans (measuring the eyeblink response byelectromyograph) or animals (measuring the startle jumping response).PPI disruptions are not only observed in Tourette Syndrome (TS)patients, but also in patients with other neuropsychiatric disorders,such as schizophrenia, ADHD, OCD, panic disorder, Huntington's disease,and nocturnal enuresis. ADHD and OCD are two common comorbidities of TS,and TS in childhood, which is a risk etiology of schizophrenia.Especially in patients with schizophrenia, PPI disruption is believed tobe a typical endophenotype of cognitive function deficit, leading tohallucination due to a flood of sensory inputs. Based on thehyperdopaminergic hypothesis, animal models with hyperdominergicactivity have been established, such as to treat animals with dopaminemimetic agents, such as methamphetamine, which facilitates dopaminerelease.

Without wishing to be bound by theory, recent studies suggest thatactivation of the cerebellum might play a role in PPI generationsince 1) PPI is enhanced in mice without cerebellar output neuron(Purkinje cell)-specific glutamate receptor subunit; and 2) PPI isassociated with decreased activation in the cerebellum.

Compounds according to the present invention, such as but not limited tocompound 6 and compound 11, LAU 159 and LAU 463 (see below and datagiven in FIG. 4) as well as their deuterated derivatives, such asDK-I-56-1, DK-I-59-1 and 1-29 (see below and data given in FIG. 6) areeffective in an animal model with sensorimotor gating deficit,reflecting METH-induced PPI impairment.

Temporomandibular Myofascial Pain and Disorders of Trigeminal Nerve

α6 subunits of GABA_(A)Rs are expressed in both neurons and satelliteglia of the trigeminal ganglia. The α6 subunit positive neuronal cellbodies in the trigeminal ganglia project axons to the temporomandibularjoint and likely to the trigeminal nucleus caudalis and upper cervicalregion (Vc-C1) and might modulate orofacial pain connected withinflammatory temporomandibular joint nociception, as well as trigeminalneuralgia and neuropathy. Without wishing to be bound by theory, chronicorofacial pain conditions are thought to arise in part (e.g.temporomandibular joint disorders) or completely (disorders oftrigeminal nerve) from damage to or pressure on the trigeminal nerve.

Rats with 30% knock down of the α6 subunit of GABA_(A) receptors intrigeminal ganglia were hypersensitive to TMJ inflammation, measured bya prolonged meal time. This suggests the α6-GABA_(A) receptor intrigeminal ganglia is important for inhibiting primary sensory afferentsin the trigeminal pathway during inflammatory orofacial nociception. Theprevalence of TMJ disorders in the United States is estimated at 4.6%and these disorders are the leading cause of chronic orofacial pain.

Moreover, α6-GABA_(A) receptors may also play an important role in otherorofacial pain disorders related to trigeminal ganglia, such astrigeminal neuralgia and trigeminal neuropathic pain. Pain in trigeminalneuralgia is sharp and electric shock-like, with a rapid onset andtermination and complete periods of remission lasting weeks to months.Trigeminal neuropathic pain is aching and throbbing, of moderateseverity, connected with dental surgery or facial trauma, and continuousin nature. There are unmet needs in the management of trigeminaldisorders. Currently, sodium channel blockers such as carbamazepinerepresent the recommended treatment for trigeminal neuralgia, whilethere are no accepted on-label pharmacotherapeutic measures fortrigeminal neuropathy.

Migraine

Migraine is a complex, disabling disorder of the brain that ischaracterized by attacks of episodic, periodic paroxysmal attacks ofthrobbing pain, separated by pain-free intervals associated with nausea,vomiting, and sensory sensitivity to light, sound, and head movement.Migraine is among the most common disorders and remains one of theleading causes of disability. Although there are several anti-migrainedrugs, many migraine patients are refractory to the current therapy orintolerable to their side effects. Therefore, a novel threatment formigraine remains an unmet medical need.

Without wishing to be bound by theory, trigemino-vascular activation viaboth peripheral and central sensitizations is considered by many to bean essential neuropathogenic mechanism of migraine, a severe headachecharacterized with concurrent nausea, vomiting, and autonomicinstability. The trigemino-vascular system (TGVS), consisting of duraland superficial cortical blood vessels which are innervated byunmyelinated trigeminal afferent C fibers and myelinated Aδ fibers, isstrongly implicated in the initiation of the headache pain. The TGVStransduces peripheral sensory signals, via trigeminal ganglia (TG), tothe trigeminal nucleus caudalis (TNC) in the brainstem, whichsubsequently projects to higher-order pain centers. In addition to TNC,the brain stem trigemino-cervical complex (TCC) also includessuperficial laminar neurons of C1/2 spinal dorsal horn neurons whichreceive nociceptive inputs via dorsal root ganglia (DRG) from C1/2dorsal roots. Besides, the TNC receives several modulatory inputs fromother brain stem nuclei, including the periaqueductal gray (PAG), locusceruleus (LC), and raphe nucleus (RN).

Central sensitization of the TVGS by TNC activation, which is involvedin both nociceptive processing and cerebrovascular regulation, isthought to play a role in migraine pathophysiology. Positron emissiontomography showed early brainstem activation in a patient duringspontaneous migraine attack but not in the headache-free interval.

Importantly, trigeminal ganglia also send projections to the trigeminalnucleus caudalis (TNC) and upper cervical region (Vc-C1), the trigeminalcervical complex. Activation of the TNC plays an important role in theneuropathogenesis of migraine.

In animal models, TGVS activation can be induced by intra-cisteral(i.c.) instillation of nociceptive substances like capsaicin orautologous blood, resulting in both central and peripheral responses.The central response in the TNC, activated by glutamate released from TGcentral terminals, can be measured by expressing c-Fos protein, aneuronal activation marker. Peripheral responses, including duralvasodilation and protein extravasation, are neurogenic inflammationphenomena mediated by neuropeptides, such as substance P or calcitoningene-related peptide (CGRP), released from TG peripheral terminals, i.e.perivascular nerve endings.

Tinnitus

Tinnitus is the perception of sound in the absence of acoustic stimuli.Around 15% of the population develops the condition of chronic tinnitusthat requires medical intervention. For about 3% of those suffering fromchronic tinnitus, the distressing symptom strongly affects their qualityof life. Various drugs have developed for clinical use in treatingtinnitus, including lidocaine (antiarrythmics), clonazepam(anxiolytics), gabapentine (anticonculsant), memantine (glutamaterantagonist), and amitriptyline (antidepressant), but they have onlylimited success.

Without wishing to be bound by theory, it is thought that cochleardamage is linked with a hypothesized neural homeostasis; that is, itargues that neural activity is constantly under homeostatic regulationin order to maintain coding efficiency (or brain functions in general)despite changes in the input environment. In sensory systems, neuralpathways are hierarchically organized. Neurons in each level are drivenby the activities originated in the preceding lower centers. Aftercochlear damages, peripheral input is suppressed. Subsequently centralregulating mechanisms are activated to maintain neural homeostasis. Thismanifests itself in the form of elevated central gain, simply tocompensate for the weakened input signals. According to this model, theincreased central gain accounts well for the physiological tinnitusexperienced by nearly 70% of normal population upon sudden entrance intoa quiet room. The central gain theoretically can be achieved byadjusting the excitation-inhibition balance in the central circuitry,likely involving the descending auditory pathways. Such derangedexcitation-inhibition balance is reflected partly in altered spontaneousneural activities, sound evoked activities and tonotopic reorganizationand findings in Fos-immuno-histochemical staining and electrophysiologyhave also been reported in experimental tinnitus on animals.

Among many central auditory relays which have been implicated intinnitus, it is thought that the dorsal cochlear nucleus (DCN) plays arole in triggering the tinnitus because it is the first obligatory relayof auditory pathways. Evidence also demonstrated that the decreases ofGABA inhibition are related to the elevation in spontaneous and evokedactivities at the DCN of loud-noise induced animals. The α6-subunit ofthe gamma-aminobutyric acid A (GABA_(A)) receptor has been found in thegranule cells of dorsal cochlear nucleus. The spontaneous activities ofgranule cells indicated by Fos immunoreactivities also increased at theDCN in tinnitus animals. Therefore, it is thought that the applicationof specific agonists of α6-subunit of GABA_(A) receptor can reduce theenhancement of neural activities indicated by the decrement ofspontaneous and sound evoked responses recorded from the dorsal cochlearnucleus and auditory cortex, Fos-immunoreactivities at the DCN, and thetinnitus related behavioral index of pre-pulse inhibition.

Depression

In addition, α6-GABA_(A) receptors are widely distributed throughout thebrain, although with a much lower concentration than that found in thegranule cells of cerebellum (Allen brain atlas). It is thought that lowabundance receptors exhibit quite specific and important functions inthe brain by modulating only those neurons on which they are located.

Since linkage studies indicate that the gene for α6-subunits isassociated with female patients with mood disorders, α6-containingreceptors might also have some beneficial effects in mood disorders suchas depression. Experiments are underway investigating whether compoundsin this patent application are able to ameliorate symptoms of depressionin several animal models of depression. The planned experiments includethe forced swim test and tail suspension tests in C57BL/6N mice, in thedesign of acute and also repeated (2 weeks) treatment; as well assucrose preference test and social interaction test in C57BL/6N micesubjected to the model of chronic social defeat stress-induced decreasein these forms of behavior. The elicited changes in behavior mimicdepression-like symptoms and could be tried to be prevented by arepeated (2 weeks) treatment with the examined compounds or citalopramused as a positive control.

The following examples further describe the present invention withoutlimitation.

EXAMPLES Example 1 Synthesis of Compounds

General Procedure A:

Exemplary compounds are shown in FIG. 23.

A substituted aniline (1 mmol) is mixed thoroughly with DEEMM (1 mmol)and heated under argon to 120° C., at which point ethanol is distilledand removed from the reaction vessel. After heating for 2-3 hours andTLC (30% EtOAc in hexanes) shows complete consumption of the startinganiline, diphenyl ether is added and the reaction vessel is heated to250° C. for 2-3 hours. After complete cyclization to the desiredquinoline the reaction vessel is cooled to RT, hexanes are added, andthe material is filtered. No further purification is performed on thismaterial.

To the quinoline (1 mmol) is added POCl3 (10 Eq.) and the mixture isstirred at an appropriate temperature to give the chlorinated quinoline.The chlorinated quinoline is further purified by flash columnchromatography (20% EtOAc in hexanes) generally giving a white/off-whitesolid.

The chlorinated quinoline (1 mmol) is mixed with xylene, triethylamine(2.5 mmol), and an appropriate hydrazine hydrochloride salt (1.5 mmol)and heated to reflux for 3-24 hours. When the reaction is complete byTLC (10% MeOH in EtOAc) the mixture is cooled to 0° C., filtered, andwashed with copious amounts of water. The solid is then washed withhexanes and allowed to dry. The solid is then recrystallized, generallyfrom a 5:1 mixture of EtOH:water, giving fluffy yellow/orange/redmicrocrystals of suitable purity for biological testing. Furtherpurification can be accomplished by dissolving the solid in DMSO andslowly adding water and/or dissolving in a basic solution (pH>10) andslowly acidifying to pH<6.

Ethyl-4-hydroxy-7-methoxy (d3) quinolone-3-carboxylate (2). A mixture of(1) (1.26 g, 0.01 m) and diethyl ethoxy methylenemalonate (DEMM) (4.32g, 0.02 m) in 50 mL diphenyl ether were heated to 120° C. for 1 hr.ethanol formed was distilled off. The solution was then heated to245-250° C. Heating was continued for 4 hrs. The contents were cooled toroom temperature. Hexane 50 mL was added and solids collected byfiltration. The compound was washed with ethyl acetate:hexane (2:1) 50mL and dried. Yield 2.37 g, 95%. Compound is off white and has very poorsolubility. The compound was used as such for the further reaction. 1HNMR (300 MHz, DMSO) 12.123 (s, 1H), 8.489(s, 1H), 8.072-8.040(d, 1H,J=9.6), 7.018-6.996(s, 2H), 4.239-4.170(q, 2H, J=6.9), 1.301-1.254(t,3H, J=6.9), 3.876(s, 3H). 13C (75 MHz, DMSO) 165.29, 162.74, 145.27,141.24, 127.99, 121.78, 114.63, 100.58, 59.95 and 14.80. HRMS m/zcalculated for C13H11D3NO4 251.1111 found 251.1110.

4-chloro-7-methoxy (d3)quinolin-3-carboxylate (3). (2) (2.5 grams, 0.01m) was heated in neat POCl3 at 80° C. for 2 hours. The excess POCl3 wasdistilled off under reduced pressure. The residue was dissolved in 25 mLdry dichloromethane and solvent distilled off under reduced pressure.The cycle was repeated for 3 times to drive off all the HCl and POCl3.Due to unstable nature of this compound it was used as such withoutfurther purification (2.55 g, 95%). 1H NMR (500 MHz, CDCl3) 9.19 (s,1H), 8.32-8.30(d, 1H, J=10.0), 7.48 (s, 1H), 7.36-7.34(t, 1H),4.52-4.48(q, 2H, J=10), 1.301-1.49-1.46(t, 3H, J=10). 13C (75 MHz,CDCl3)164.46, 162.87, 151.28, 150.63, 143.94, 126.86, 121.73, 121.36,120.69, 107.27, 61.95, 55.14 and 14.29. HRMS m/z calculated forC13H10D3ClNO3 269.0772 found 269.0770.

A general procedure for the syntheses of 4. A mixture of4-chloro-7-methoxy(d3)quinolin-3-carboxylate (3) (0.01 mol, 0.324 g),substituted Phenylhydrazine hydrohloride (0.012 mol) and TEA (0.012m,0.12 g) in 40 mL xylene was refluxed for 4 hr. The reaction mixturewas cooled to room temperature. The precipitated compound was collectedby filtration and purified by crystallization.

Synthesis of LAU159, LAU165, and LAU463

8-Chloro-2-(3-methoxyphenyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one(LAU159). In a 8 mL-vial with magnetic stirrer and screw cap, ethyl4,8-dichloroquinoline-3-carboxylate (135 mg, 0.5 mmol, 1 equiv.),3-(methoxy)phenyl hydrazine (83 mg, 0.6 mmol, 1.2 equiv.) andtriethylamine (61 mg, 0.6 mmol, 1.2 equiv.) were dissolved in dryN,N-dimethylacetamide (3 mL). The reaction mixture was heated to 140° C.for 16 hours. After completion of the reaction evaporation of volatilesand washing of the solid residue with acetone and water afforded thepure product. Yield: 66% (0.33 mmol, 108 mg), Appearance: yellow solid,M.p.: ˜340° C., with partial decomposition above 300° C., 1H NMR (200MHz, DMSO-d6): 12.95 (bs, 1H), 8.72 (d, J=5.2 Hz, 1H), 8.15 (s, 1H),7.84-7.70 (m, 4H), 7.33 (t, J=8.1 Hz, 1H), 6.75 (d, J=8.1 Hz, 1H), 3.80(s, 3H). 13C NMR (50 MHz, DMSO-d6): δ=161.5 (s), 159.5 (s), 141.9 (s),141.0 (s), 139.5 (d), 134.2 (s), 130.6 (s), 130.2 (d), 129.5 (d), 121.6(d), 121.1 (d), 119.9 (s), 110.9 (d), 109.5 (d), 106.3 (s), 104.4 (d),55.1 (q). HR-MS: [M+H]+m/z (predicted)=326.0691, m/z(measured)=326.0688, difference=−0.92 ppm.

8-Chloro-2-(2-methoxyphenyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one(LAU165). In an 8 mL-vial with magnetic stirrer and screw cap, ethyl4,6-dichloroquinoline-3-carboxylate (135 mg, 0.5 mmol, 1 equiv),2-methoxyphenylhydrazine (83 mg, 0.6 mmol, 1.2 equiv) and triethylamine(63 mg, 0.6 mmol, 1.2 equiv) were dissolved in dry N,N-dimethylacetamide(3 mL). The reaction mixture was heated to 140° C. for 16 hours. Aftercompletion of the reaction the reaction mixture was evaporated todryness. The crude product was purified by flash-column chromatography(45 g silica 60, eluent EtOAc/MeOH 5%) Co-eluting triethylaminehydrochloride was subsequently removed by washing with water. Yield: 28%(0.14 mmol, 46 mg) (28%). Appearance: yellow solid. TLC: 0.07(EtOAc/MeOH 10%). M.p. 310-313° C. with partial decomposition. 1H-NMR(200 MHz, DMSO-d₆) δ=3.73 (s, 3H), 7.03 (t, J=7.5 Hz, 1H), 7.16 (d,J=8.2 Hz, 1H), 7.29-7.45 (m, 2H), 7.62-7.73 (m, 2H), 8.01 (d, J=1.6 Hz,1H), 8.65 (s, 1H), 12.78 (s, 1H). 13C-NMR (50 MHz, DMSO-d₆) δ=55.6 (q),105.3 (s), 112.5 (d), 120.2 (d), 120.3 (s), 120.9 (d), 121.4 (d), 127.7(s), 129.3 (d), 129.4 (d), 129.7 (d), 130.3 (s), 134.0 (s), 139.1 (d),141.4 (s), 155.1 (s), 161.6 (s). HR-ESI-MS: m/z 326.0678 [M+H]⁺ (calcd326.0691, diff-3.99 ppm)

7-Bromo-2-(4-methoxyphenyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one(LAU463). Ethyl 7-bromo-4-chloroquinoline-3-carboxylate (200 mg, 0.64mmol, 1 equiv.) and (4-methoxyphenyl)hydrazine (110 mg, 0.80 mmol, 1.2equiv.) were dissolved in 5 mL dimethylacetamide. The reaction wascarefully purged with argon several times, triethylamine (1 equiv.) wasadded and the reaction was heated to 140° C. for 24 hours. The solventwas removed via Kugelrohr distillation of the crude mixture. Washing ofthe residue with acetone gave the product as an orange-yellow solid.Yield: 51% (0.33 mmol, 121 mg), Appearance: orange-yellow solid, TLC:0.45 (EtOAc/MeOH 20%), M.p.: >330° C., 1H NMR (400 MHz, DMSO-d6) δ 12.78(s, 1H), 8.74 (d, J=5.8 Hz, 1H), 8.13 (d, J=8.5 Hz, 1H), 8.06 (d, J=8.6Hz, 2H), 7.89 (d, J=2.0 Hz, 1H), 7.69 (dd, J=8.5, 2.0 Hz, 1H), 7.02 (d,J=8.6 Hz, 2H), 3.79 (s, 3H). 13C NMR (101 MHz, DMSO) δ 161.3 (s), 156.5(s), 142.4 (s), 140.1 (d), 136.9 (s), 133.8 (s), 129.7(d), 124.5 (d),123.0 (s), 122.2 (d), 120.9 (d), 118.2 (s), 114.3 (d), 107.2 (s), 55.7(q)

7-Methoxy-2-(pyrazin-2-yl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one(DCBS126). In a 8 mL vial with magnetic stirrer and screw cap, ethyl4-chloro-7-methoxyquinoline-3-carboxylate (70 mg, 0.26 mmol, 1 equiv.)and 2-hydrazinopyrazine (32 mg, 0.29 mmol, 1.1 equiv.) were dispersed in1.5 mL ethanol, triethylamine (40 μL, 0.29 mmol, 1.1 eq.) was added andthe reaction mixture was heated to reflux under argon atmosphere. After20 h the reaction mixture was rinsed with 4 mL water, filtered and theprecipitate was washed with 15 mL EtOAc/PE (1/1). The yellow solid wasdried under reduced pressure to give the desired product. Yield: 58%(0.15 mmol, 45 mg), Appearance: yellow solid, TLC: 0.38 (10% MeOH inCH2C12), M.p.: >300° C., 1H NMR (400 MHz, DMSO-d6) δ 3.88 (s, 3H),7.16-7.23 (m, 2H), 8.13 (dd, J=8.5, 0.8 Hz, 1H), 8.44 (d, J=2.5 Hz, 1H),8.56 (dd, J=2.5, 1.5 Hz, 1H), 8.75 (s, 1H), 9.51 (d, J=1.4 Hz, 1H),12.74 (br s, 1H).13C NMR (101 MHz, DMSO-d6) δ 55.6 (q), 102.1 (d), 105.0(s), 112.2 (s), 115.5 (d), 123.9 (d), 136.6 (d), 137.3 (s), 140.0 (d),140.1 (d), 142.8 (d), 144.9 (s), 148.0 (s), 160.8 (s), 162.4 (s). HR-MS:Calc.[M+H]+ m/z (predicted)=294.0992, m/z (measured)=294.0992,difference=0.00 ppm.

N-(4-Hydroxyphenyl)acetamide [DK-I-2-1]. To a mixture of 4-aminophenol(50.0 g, 458.2 mmol) and tetrahydrofuran (200 mL) acetic anhydride (49.1g, 481.1 mmol) was added dropwise over 30 min while keeping thetemperature below 50° C. The reaction mixture was then stirred for 30min at 50° C. and then cooled to rt. The reaction mixture was thendiluted with hexanes (200 mL) to precipitate the product. After stirringfor 1 h, the solid product was filtered and washed twice with hexanes(50 mL×2). The solid was dried to afford the product as a whitecrystalline solid DK-I-2-1 (62.7 g, 90.0%): mp 170-171° C.; ¹H NMR (300MHz, DMSO) δ 9.64 (s, 1H), 9.13 (s, 1H), 7.34 (d, J=8.8 Hz, 2H), 6.67(d, J=8.8 Hz, 2H), 1.98 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 167.95,153.57, 131.49, 121.26, 115.44, 24.19; HRMS m/z calculated for C8H10NO2(M+H)⁺ 152.0711 found 152.15.

N-(3-Hydroxyphenyl)acetamide [DK-I-3-1]. To a mixture of 3-aminophenol(25.0 g, 229.1 mmol) and tetrahydrofuran (100 mL) acetic anhydride (24.5g, 240.5 mmol) was added dropwise over 30 min while keeping thetemperature below 50° C. The reaction mixture was then stirred for 30min at 50° C. and then cooled to room temperature. The reaction mixturewas then diluted with hexanes (100 mL) to precipitate the product. Afterstirring for 1 h, the solid product was filtered and washed twice withhexanes (25 mL×2). The solid was dried to afford the product as a whitecrystalline solid DK-I-3-1 (33.2 g, 96.0%): mp 145-148° C.; ¹H NMR (300MHz, DMSO) δ 9.77 (s, 1H), 9.32 (s, 1H), 7.18 (s, 1H), 7.04 (t, J=8.0Hz, 1H), 6.92 (d, J=8.1 Hz, 1H), 6.42 (dd, J=7.9, 2.1 Hz, 1H), 2.01 (s,3H); ¹³C NMR (75 MHz, DMSO) δ 168.60, 158.01, 140.81, 129.72, 110.55,110.18, 106.60, 24.50; HRMS m/z calculated for C8H10NO2 (M+H)⁺ 152.0711found 152.15.

N-(2-Hydroxyphenyl)acetamide [DK-I-30-1]. To a mixture of 2-aminophenol(25.0 g, 229.1 mmol) and tetrahydrofuran (100 mL) acetic anhydride (24.5g, 240.5, mmol) was added dropwise over 30 min while keeping thetemperature below 50° C. The reaction mixture was then stirred for 30min at 50° C. and then cooled to rt. The reaction mixture was thendiluted with hexanes (100 mL) to precipitate the product. After stirringfor 1 h, the solid product was filtered and washed twice with hexanes(25 mL×2). The solid was dried to afford the product as a light brownsolid DK-I-30-1 (33.1 g, 95.7%): mp 211-213° C.; ¹H NMR (300 MHz, DMSO)δ 9.75 (s, 1H), 9.31 (s, 1H), 7.67 (d, J=7.7 Hz, 1H), 6.85 (ddd, J=33.6,14.6, 7.2 Hz, 3H), 2.10 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 169.44,148.34, 126.88, 125.08, 122.80, 119.40, 116.38, 24.05; HRMS m/zcalculated for C8H10NO2 (M+H)⁺ 152.0711 found 152.15.

N-(4-Methoxy-d3-phenyl)acetamide [DK-I-6-1]. To a mixture ofN-(4-hydroxyphenyl)acetamide DK-I-2-1 (62.0 g, 410.1 mmol), potassiumcarbonate (113.4 g, 615.2 mmol) and acetone (230 mL) methyl iodide (D3)(100 g, 689.8 mmol) was added dropwise over 30 min. The reaction mixturewas then stirred for 24 h at 20-25° C. The reaction mixture was thendiluted with ethyl acetate (300 mL) and water (300 mL). The resultingbiphasic mixture was allowed to stand for 15 min and the layers wereseparated. The aqueous layer was extracted with ethyl acetate (200 mL)and then the combined organic layers were washed with 10% potassiumcarbonate solution (200 mL). The organic layer was then dried overmagnesium sulfate. The solvents were then removed by evaporation on arotovap and the product residue was slurried with hexanes (200 mL). Thesolid product was then filtered and washed twice with hexanes (50 mL×2).The solid was dried to afford the product as an off-white solid DK-I-6-1(71.7 g, 99%): mp 125-126° C.; ¹H NMR (300 MHz, DMSO) δ 9.77 (s, 1H),7.48 (d, J=9.0 Hz, 2H), 6.85 (d, J=9.0 Hz, 2H), 3.38 (s, 3H), 2.00 (s,3H); ¹³C NMR (75 MHz, DMSO) δ 168.20, 155.48, 132.94, 121.01, 114.21,24.23; HRMS m/z calculated for C9H9D3NO2 (M+H)⁺ 169.1054 found 169.20.

N-(3-Methoxy-d3-phenyl)acetamide [DK-I-8-1]. To a mixture ofN-(3-hydroxyphenyl)acetamide DK-I-3-1 (35.0 g, 231.5 mmol), potassiumcarbonate (64.0 g, 463.1 mmol) and acetone (140 mL) methyl iodide (D3)(50.3 g, 347.3 mmol) was added dropwise over 30 min. The reactionmixture was then stirred for 24 h at 20-25° C. The reaction mixture wasthen diluted with ethyl acetate (150 mL) and water (150 mL). Theresulting biphasic mixture was allowed to stand for 15 min and thelayers were separated. The aqueous layer was extracted with ethylacetate (100 mL) and then the combined organic layers were washed with10% potassium carbonate solution (100 mL). The organic layer was thendried over magnesium sulfate. The solvents were then removed byevaporation on a rotovap and the product residue was slurried withhexanes (100 mL). The solid product was then filtered and washed twicewith hexanes (50 mL×2). The solid was dried to afford the product as anoff-white solid DK-I-8-1 (38.9 g, 99%): mp 89-91° C;¹H NMR (300 MHz,DMSO) δ 9.89 (s, 1H), 7.27 (s, 1H), 7.18 (t, J=8.1 Hz, 1H), 7.10 (d,J=8.2 Hz, 1H), 6.60 (dd, J=7.8, 2.0 Hz, 1H), 2.03 (s, 3H); ¹³C NMR (75MHz, DMSO) δ 168.76, 159.93, 140.96, 129.89, 111.71, 108.76, 105.30,24.53; HRMS m/z calculated for C9H9D3NO2 (M+H)⁺ 169.1054 found 169.15.

N-(2-Methoxy-d3-phenyl)acetamide [DK-I-31-1]. To a mixture ofN-(2-hydroxyphenyl)acetamide DK-I-30-1 (30.0 g, 198.5 mmol), potassiumcarbonate (54.9 g, 396.9 mmol) and acetone (140 mL) methyl iodide (D3)(50.3 g, 347.3 mmol) was added dropwise over 30 min. The reactionmixture was then stirred for 24 h at 20-25° C. The reaction mixture wasthen diluted with ethyl acetate (150 mL) and water (150 mL). Theresulting biphasic mixture was allowed to stand for 15 min and thelayers were separated. The aqueous layer was extracted with ethylacetate (100 mL) and then the combined organic layers were washed with10% potassium carbonate solution (100 mL). The organic layer was thendried over magnesium sulfate. The solvents were then removed byevaporation on a rotovap and the product residue was slurried withhexanes (100 mL). The solid product was then filtered and washed twicewith hexanes (50 mL×2). The solid was dried to afford the product as anoff-white solid DK-I-31-1 (31.9 g, 99%): mp 82-83° C.; ¹H NMR (300 MHz,DMSO) δ 9.10 (s, 1H), 7.93 (d, J=7.8 Hz, 1H), 7.22-6.97 (m, 2H),6.97-6.79 (m, 1H), 2.08 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 168.87,149.98, 127.87, 124.63, 122.45, 120.57, 111.50, 24.30; HRMS m/zcalculated for C9H9D3NO2 (M+H)⁺ 169.1054 found 169.15.

4-Methoxy-d3-aniline [DK-I-67-1]. A mixture ofN-(4-methoxy-d3-phenyl)acetamide DK-I-6-1 (20.0 g, 118.9 mmol), 12 Mhydrochloric acid (20 mL, 240 mmol), and water (60 mL) was heated at90-95° C. for 2 h. The reaction mixture was then cooled to 20-25° C. andthe pH was adjusted to 14 with a solution of sodium hydroxide (20 g, 500mmol) and water (20 mL). The product was then extracted from the aqueouslayer four times with dichloromethane (50 mL×4). The combined organiclayers were then dried over magnesium sulfate. Evaporation of thesolvents on a rotovap afforded the product as a dark orange oilDK-I-67-1 (14.4 g, 96%): ¹H NMR (300 MHz, DMSO) δ 5.75-5.62 (m, 2H),5.62-5.47 (m, 2H), 3.50 (s, 2H); ¹³C NMR (75 MHz, DMSO) δ 150.34,141.51, 114.62, 113.89; HRMS m/z calculated for C7H7D3NO (M+H)⁺ 127.0948found 127.25.

3-Methoxy-d3-aniline [DK-I-41-1]. A mixture ofN-(3-methoxy-d3-phenyl)acetamide DK-I-8-1 (20.0 g, 118.9 mmol), 12 Mhydrochloric acid (20 mL, 240 mmol), and water (60 mL) was heated at90-95° C. for 2 h. The reaction mixture was then cooled to 20-25° C. andthe pH was adjusted to 14 with a solution of sodium hydroxide (20 g, 500mmol) and water (20 mL). The product was then extracted from the aqueouslayer four times with dichloromethane (50 mL×4). The combined organiclayers were then dried over magnesium sulfate. Evaporation of thesolvents on a rotovap afforded the product as a golden yellow oilDK-I-41-1 (13.5 g, 90%): ¹H NMR (300 MHz, CDCl₃) δ 7.10 (t, J=8.0 Hz,1H), 6.35 (dddd, J=11.9, 11.2, 3.4, 2.0 Hz, 3H), 4.00 (s, 2H); ¹³C NMR(75 MHz, CDCl₃) δ 160.78, 147.54, 130.16, 108.14, 104.22, 101.28; HRMSm/z calculated for C7H6D3NO (M+H)⁺ 127.0948 found 127.25.

Ethyl-6-chloro-4-hydroxyquinoline-3-carboxylate [DK-I-34-1]. A mixtureof 4-chloroaniline (45.5 g, 356.7 mmol), diethyl ethoxymethylenemalonate(80.9 g, 374.1 mmol) and diphenyl ether (200 mL) was slowly heated to230° C. The evolved ethanol was collected in a Dean-Stark trap. Once theethanol formation ceased, the reaction mixture was heated for anadditional 30 min at 230° C. The reaction mixture was then cooled to 80°C. and diluted with ethanol (200 mL). Upon cooling to 20-25° C. thesolid product was collected by filtration and washed twice with ethanol(50 mL×2) and twice with hexanes (50 mL×2). The solid was dried toafford the product as an off-white crystalline solid DK-I-34-1 (85.1 g,95%): ¹H NMR (300 MHz, TFA) δ 11.66 (s, 1H), 9.32 (d, J=4.5 Hz, 1H),8.62 (d, J=2.5 Hz, 1H), 8.12 (d, J=13.0 Hz, 2H), 4.82-4.55 (m, 2H), 1.53(dd, J=11.8, 7.0 Hz, 3H); ¹³C NMR (75 MHz, TFA) δ 172.51, 167.19,144.95, 138.35, 137.62, 137.58, 123.58, 121.35, 120.82, 105.30, 64.70,11.96; HRMS m/z calculated for C12H11ClNO3 (M+H)⁺ 252.0427 found 252.10.

Ethyl-4-hydroxy-7-methoxyquinoline-3-carboxylate [DK-I-39-1]. A mixtureof 3-methoxyaniline (50.0 g, 406.0 mmol), diethylethoxymethylenemalonate (87.8 g, 406.0 mmol) and diphenyl ether (200 mL)was slowly heated to 230° C. The evolved ethanol was collected in aDean-Stark trap. Once the ethanol formation ceased, the reaction mixturewas heated for an additional 30 min at 230° C. The reaction mixture wasthen cooled to 80° C. and diluted with ethanol (200 mL). Upon cooling to20-25° C. the solid product was collected by filtration and washed twicewith ethanol (50 mL×2) and twice with hexanes (50 mL×2). The solid wasdried to afford the product as a light brown solid DK-I-39-1 (37.1 g,37%): ¹H NMR (300 MHz, TFA) δ 11.63 (s, 1H), 9.22 (d, J=6.3 Hz, 1H),8.56 (dd, J=9.1, 6.7 Hz, 1H), 7.66-7.54 (m, 1H), 7.47 (d, J=4.2 Hz, 1H),4.69 (dd, J=13.8, 6.9 Hz, 2H), 4.13 (d, J=6.4 Hz, 3H), 1.57 (q, J=6.8Hz, 3H); ¹³C NMR (75 MHz, TFA) δ 171.88, 167.91, 167.62, 144.49, 142.43,126.28, 121.92, 114.05, 103.81, 99.24, 64.28, 55.45, 12.00; HRMS m/zcalculated for C13H14NO4 (M+H)⁺ 248.0923 found 248.15.

Ethyl-7-bromo-4-hydroxyquinoline-3-carboxylate [DK-I-49-1]. A mixture of3-bromoaniline (8.7 g, 58.1 mmol), diethyl ethoxymethylenemalonate (10.9g, 58.1 mmol) and diphenyl ether (40 mL) was slowly heated to 230° C.The evolved ethanol was collected in a Dean-Stark trap. Once the ethanolformation ceased, the reaction mixture was heated for an additional 30min at 230° C. The reaction mixture was then cooled to 80° C. anddiluted with ethanol (40 mL). Upon cooling to 20-25° C. the solidproduct was collected by filtration and washed twice with ethanol (10mL×2) and twice with hexanes (10 mL×2). The solid was dried to affordthe product as a light brown solid DK-I-49-1 (11.5 g, 77%): ¹H NMR (300MHz, TFA) δ 11.64 (s, 1H), 9.38 (s, 1H), 8.57 (d, J=8.9 Hz, 1H), 8.43(s, 1H), 8.15 (d, J=8.9 Hz, 1H), 4.75 (q, J=7.1 Hz, 2H), 1.60 (t, J=7.2Hz, 3H); ¹³C NMR (75 MHz, TFA) δ 173.48, 167.33, 145.75, 139.71, 134.19,134.06, 125.60, 122.70, 118.57, 105.08, 64.74, 12.01; HRMS m/zcalculated for C12H10BrNO3 (M+H)⁺ 295.9922 found 296.05.

Ethyl-4-hydroxy-7-methoxy-d3-quinoline-3-carboxylate [DK-I-54-1]. Amixture of 3-methoxy-d3-aniline DK-I-41-1 (10 g, 81.2 mmol), diethylethoxymethylenemalonate (21.1 g, 97.4 mmol) and diphenyl ether (100 mL)was slowly heated to 230° C. The evolved ethanol was collected in aDean-Stark trap. Once the ethanol formation ceased, the reaction mixturewas heated for an additional 30 min at 230° C. The reaction mixture wasthen cooled to 80° C. and diluted with hexanes (100 mL). Upon cooling to20-25° C. the solid product was collected by filtration and washed twicewith hexanes (50 mL×2). The solid was dried to afford the product as abrown solid DK-I-54-1 (13.0 g, 64%): ¹H NMR (300 MHz, TFA) δ 11.64 (s,1H), 9.23 (s, 1H), 8.57 (d, J=9.3 Hz, 1H), 7.59 (dd, J=9.4, 2.3 Hz, 1H),7.48 (d, J=2.2 Hz, 1H), 4.71 (q, J=7.2 Hz, 2H), 1.58 (t, J=7.2 Hz, 3H);¹³C NMR (75 MHz, TFA) δ 171.89, 167.92, 167.64, 144.50, 142.44, 126.28,121.93, 114.05, 103.81, 99.25, 64.29, 12.01; HRMS m/z calculated forC13H11D3NO4 (M+H)⁺ 251.1109 found 251.20.

Ethyl-4-hydroxy-6-methoxy-d3-quinoline-3-carboxylate [DK-I-70-1]. Amixture of 4-methoxy-d3-aniline DK-I-67-1 (10 g, 81.2 mmol), diethylethoxymethylenemalonate (21.1 g, 97.4 mmol) and diphenyl ether (100 mL)was slowly heated to 230° C. The evolved ethanol was collected in aDean-Stark trap. Once the ethanol formation ceased, the reaction mixturewas heated for an additional 30 min at 230° C. The reaction mixture wasthen cooled to 80° C. and diluted with hexanes (100 mL). Upon cooling to20-25° C. the solid product was collected by filtration and washed twicewith hexanes (50 mL×2). The solid was dried to afford the product as alight brown solid DK-I-70-1 (9.9 g, 49%). ¹H NMR (300 MHz, TFA) δ 11.66(s, 1H), 9.15 (s, 1H), 8.05 (d, J=9.2 Hz, 1H), 7.97-7.74 (m, 2H), 4.67(q, J=7.1 Hz, 2H), 1.67-1.39 (m, 3H); ¹³C NMR (75 MHz, TFA) δ 171.68,167.54, 160.89, 141.86, 134.62, 129.91, 121.73, 121.28, 104.58, 102.17,64.41, 11.97; HRMS m/z calculated for C13H11D3NO4 (M+H)⁺ 251.1109 found251.20.

Ethyl 6-bromo-8-fluoro-4-hydroxyquinoline-3-carboxylate [MM-I-01].3-Bromo-5-fluoroaniline (10 g, 52.6 mmol) was heated with diethylethoxymethylene malonate (11.2 mL, 55.3 mmol) at 125° C. After heatingfor 2 h, downtherm A (50 mL) was added and the mixture was heated up to255° C. for more 2 h. The reaction was brought to room temperature anddiluted with hexane (50 mL). The mixture was stirred for 5 min. Theprecipitate was filtered and washed with hexane to yield the product asa brown colored solid MM-I-01 (13.60 g, 82%): mp 285-286° C.; 1H NMR(300 MHz, DMSO) δ 12.65 (s, 1H; H-11), 8.40 (s, 1H; H-8), 8.04 (s, 1H;H-6), 8.00 (dd, J=10.1, 2.0 Hz, 1H; H-2), 4.23 (q, J=7.1 Hz, 2H; H-18),1.28 (t, J=7.1 Hz, 3H; H-17); HRMS m/z calculated for C12H9NO3FBr313.9823 found 313.9833.

Ethyl 4-hydroxy-7-(trifluoromethyl)quinoline-3-carboxylate [MM-I-04].3-(Trifluoromethyl)aniline (10 g, 62.1 mmol) was heated with diethylethoxymethylene malonate (12.6 mL, 62 mmol) at 125° C. for 1 h. Then,downtherm A (50 mL) was added and the mixture was heated up to 255° C.for 2.5 h. After heating, the reaction was brought to room temperatureand diluted with hexane (50 mL). The mixture was stirred for 5 min. Theprecipitated was filtered and washed with hexane to provide the productas a white colored solid MM-I-04 (16.51 g, 93%): mp 340-341° C.; 1H NMR(300 MHz, DMSO) δ 12.51 (s, 1H; H-11), 8.70 (s, 1H; H-8), 8.35 (d, J=8.3Hz, 1H; H-6), 8.00 (s, 1H; H-3), 7.72 (d, J=8.1 Hz, 1H; H-1), 4.24 (q,J=14.3, 7.1 Hz, 2H; H-20), 1.29 (t, J=7.0 Hz, 3H; H-19); HRMS m/zcalculated for C13H10NO3F3 286.0686 found 286.0691.

Ethyl-4,6-dichloroquinoline-3-carboxylate [DK-I-35-1]. A mixture ofethyl-6-chloro-4-hydroxyquinoline-3-carboxylate DK-I-34-1 (85.1 g, 338.1mmol), N,N-dimethylformamide (1.0 mL, 12.9 mmol), and dichloromethane(640 mL) was heated to 35-40° C. Oxalyl chloride (47.2 g, 371.9 mmol)was added dropwise to the reaction mixture over 30 min. The reactionmixture was then heated for 6 h at reflux (38-40° C.). The resultingpale yellow solution was then cooled to 20-25° C. The reaction mixturewas then neutralized by slowly adding a 25% solution of potassiumcarbonate (75 g) in water (300 mL). The layers were then separated andthe aqueous layers extracted with dichloromethane (200 mL). The combinedorganic layers were then washed with a 25% solution of potassiumcarbonate (50 g) in water (200 mL). The combined organic layers werethen dried over magnesium sulfate. The solvents were then removed byevaporation on a rotovap and the product residue was slurried withhexanes (200 mL). The solid product was then filtered and washed twicewith hexanes (50 mL×2). The solid was dried to afford the product as anoff-white solid DK-I-35-1 (81.9 g, 90%): ¹H NMR (300 MHz, DMSO) δ 9.13(s, 1H), 8.30 (d, J=2.2 Hz, 1H), 8.14 (d, J=9.0 Hz, 1H), 7.97 (dd,J=9.0, 2.3 Hz, 1H), 4.44 (q, J=7.1 Hz, 2H), 1.39 (t, J=7.1 Hz, 3H); ¹³CNMR (75 MHz, DMSO) δ 164.01, 150.53, 147.73, 141.04, 134.30, 133.34,132.20, 126.53, 124.37, 124.08, 62.59; HRMS m/z calculated forC12H10Cl2NO2 (M+H)⁺ 270.0088 found 270.10.

Ethyl-4-chloro-7-methoxyquinoline-3-carboxylate [DK-I-40-1]. A mixtureof ethyl-4-hydroxy-7-methoxyquinoline-3-carboxylate DK-I-39-1 (37.1 g,150.0 mmol), N,N-dimethylformamide (0.5 mL, 6.5 mmol), anddichloromethane (150 mL) was heated to 35-40° C. Oxalyl chloride (20.9g, 165.0 mmol) was added dropwise to the reaction mixture over 30 min.The reaction mixture was then heated for 2 h at reflux (38-40° C.). Theresulting brown solution was then cooled to 20-25° C. The reactionmixture was diluted with dichloromethane (150 mL) and then neutralizedby slowly adding a 25% solution of potassium carbonate (75 g) in water(300 mL). The layers were then separated and the aqueous layersextracted with dichloromethane (100 mL). The combined organic layerswere then washed with a 25% solution of potassium carbonate (75 g) inwater (300 mL). The combined organic layers were then dried overmagnesium sulfate. The solvents were then removed by evaporation on arotovap and the product residue was slurried with hexanes (200 mL). Thesolid product was then filtered and washed twice with hexanes (50 mL×2).The solid was dried to afford the product as an off-white solidDK-I-40-1 (36.3 g, 91%): ¹H NMR (300 MHz, DMSO) δ 9.08 (s, 1H), 8.25 (d,J=9.2 Hz, 1H), 7.57-7.37 (m, 2H), 4.41 (q, J=7.1 Hz, 2H), 3.98 (s, 3H),1.38 (t, J=7.1 Hz, 3H); ¹³C NMR (75 MHz, DMSO) δ 164.32, 162.83, 151.62,150.81, 142.02, 126.85, 122.12, 121.11, 120.56, 108.36, 62.15, 56.45,14.49; HRMS m/z calculated for C13H13ClNO3 (M+H)⁺ 266.0584 found 266.15.

Ethyl-7-bromo-4-chloroquinoline-3-carboxylate [DK-I-54-1]. A mixture ofethyl-7-bromo-4-hydroxyquinoline-3-carboxylate DK-I-49-1 (11.0 g, 37.1mmol), N,N-dimethylformamide (0.1 mL, 1.3 mmol), and dichloromethane (50mL) was heated to 35-40° C. Oxalyl chloride (5.2 g, 40.9 mmol) was addeddropwise to the reaction mixture over 30 min. The reaction mixture wasthen heated for 1 h at reflux (38-4 0° C.). The resulting brown solutionwas then cooled to 20-25° C. The reaction mixture was diluted withdichloromethane (150 mL) and then neutralized by slowly adding a 25%solution of potassium carbonate (12.5 g) in water (50 mL). The layerswere then separated and the aqueous layers extracted withdichloromethane (50 mL). The combined organic layers were then washedwith a 25% solution of potassium carbonate (12.5 g) in water (50 mL).The combined organic layers were then dried over magnesium sulfate. Thesolvents were then removed by evaporation on a rotovap and the productresidue was slurried with hexanes (50 mL). The solid product was thenfiltered and washed twice with hexanes (25 mL×2). The solid was dried toafford the product as an off-white solid DK-I-54-1 (7.2 g, 61%): ¹H NMR(300 MHz, DMSO) δ 9.15 (s, 1H), 8.36 (d, J=1.9 Hz, 1H), 8.28 (d, J=9.0Hz, 1H), 7.98 (dd, J=9.0, 1.9 Hz, 1H), 4.44 (q, J=7.1 Hz, 2H), 1.39 (t,J=7.1 Hz, 3H); ¹³C NMR (75 MHz, DMSO) δ 164.04, 151.40, 149.74, 142.34,132.67, 131.88, 127.50, 126.64, 124.70, 123.96, 62.55, 14.46; HRMS m/zcalculated for C12H10BrClNO2(M+H)⁺ 313.9583 found 314.05.

Ethyl-4-chloro-7-methoxy-d3-quinoline-3-carboxylate [DK-I-57-1]. Amixture of ethyl-4-hydroxy-7-methoxy-d3-quinoline-3-carboxylateDK-I-54-1 (13.0 g, 51.9 mmol), phosphorus oxychloride (8.8 g, 57.1 mmol)and toluene (52 mL) was heated to 80-90° C. The reaction mixture wasthen held for 1 h at 80-90° C.). The resulting brown solution was thencooled to 20-25° C. The reaction mixture was then diluted with hexanes(50 mL). The solids were collected by filtration and washed twice withhexanes (50 mL each). The solids were then dissolved in dichloromethane(100 mL) and then neutralized by slowly adding a 25% solution ofpotassium carbonate (12.5 g) in water (50 mL). The layers were thenseparated and the aqueous layers extracted with dichloromethane (50 mL).The combined organic layers were then washed with a 25% solution ofpotassium carbonate (12.5 g) in water (50 mL). The combined organiclayers were then dried over magnesium sulfate. The solvents were thenremoved by evaporation on a rotovap and the product residue was slurriedwith hexanes (50 mL). The solid product was then filtered and washedtwice with hexanes (25 mL×2). The solid was dried to afford the productas an off-white solid DK-I-57-1 (11.5 g, 82%): ¹H NMR (300 MHz, DMSO) δ9.07 (s, 1H), 8.23 (d, J=9.2 Hz, 1H), 7.54-7.38 (m, 2H), 4.40 (q, J=7.1Hz, 2H), 1.37 (t, J=7.1 Hz, 3H); ¹³C NMR (75 MHz, DMSO) δ 164.29,162.85, 151.53, 150.75, 142.41, 126.84, 122.11, 121.07, 120.56, 108.27,62.15, 14.49; HRMS m/z calculated for C13H10D3ClNO3 (M+H)⁺ 269.0770found 269.15.

Ethyl-4-chloro-6-methoxy-d3-quinoline-3-carboxylate [DK-I-73-2]. Amixture of ethyl-4-hydroxy-6-methoxy-d3-quinoline-3-carboxylateDK-I-70-1 (10.0 g, 40.0 mmol), phosphorus oxychloride (6.7 g, 44.0 mmol)and toluene (40 mL) was heated to 80-90° C. The reaction mixture wasthen held for 1 h at 80-90° C.). The resulting brown solution was thencooled to 20-25° C. The reaction mixture was then diluted with hexanes(40 mL). The solids were collected by filtration and washed twice withhexanes (20 mL each). The solids were then dissolved in dichloromethane(100 mL) and then neutralized by slowly adding a 25% solution ofpotassium carbonate (10 g) in water (40 mL). The layers were thenseparated and the aqueous layers extracted with dichloromethane (50 mL).The combined organic layers were then washed with a 25% solution ofpotassium carbonate (10 g) in water (40 mL). The combined organic layerswere then dried over magnesium sulfate. The solvents were then removedby evaporation on a rotovap and the product residue was slurried withhexanes (50 mL). The solid product was then filtered and washed twicewith hexanes (25 mL×2). The solid was dried to afford the product as anoff-white solid DK-I-73-2 (8.5 g, 79%): ¹H NMR (300 MHz, DMSO) δ 8.95(s, 1H), 8.03 (d, J=9.1 Hz, 1H), 7.67-7.45 (m, 2H), 4.42 (q, J=7.1 Hz,2H), 1.38 (t, J=7.1 Hz, 3H); ¹³C NMR (75 MHz, DMSO) δ 164.49, 159.57,147.25, 145.36, 139.99, 131.67, 126.91, 125.20, 123.87, 102.96, 62.36,14.47; HRMS m/z calculated for C13H10D3ClNO3 (M+H)⁺ 269.0770 found269.15.

Ethyl 6-bromo-4-chloro-8-fluoroquinoline-3-carboxylate [MM-I-02]. TheEthyl 6-bromo-8-fluoro-4-hydroxyquinoline-3-carboxylate MM-I-01 (1 g,3.2 mmol) was placed in a flask with POCl3 (4 mL). The mixture washeated at 70° C. for 3 h. The excess of POCl3 was evaporated by reducedpressure and the remaining oil was quenched with saturated solution ofNaHCO3. Then, the aqueous solution was extracted with CH2Cl2 (3×50 mL)and the combined organic layers were dried (Na2SO4). The solvent wasremoved under reduce pressure and the residue was purified by silica gelchromatography to give the compound as a white solid MM-I-02 (0.79 g,75%): 1H NMR (300 MHz, CDCl3) δ 9.23 (s, 1H; H-8), 8.40 (s, 1H; H-6),7.70 (dd, J=9.1, 1.9 Hz, 1H; H-2), 4.54 (q, J=7.1 Hz, 2H; H-18), 1.49(t, J=7.1 Hz, 3H; H-17). HRMS m/z calculated for C12H8NO2FClBr 331.9484found 331.9487.

Ethyl 4-chloro-7-(trifluoromethyl)quinoline-3-carboxylate [MM-I-05]. Thereaction was performed following the same procedure as for MM-I-02except the reaction was performed in bigger scale. Ethyl4-hydroxy-7-(trifluoromethyl)quinoline-3-carboxylate MM-I-04 (5 g, 18mmol) was heated with POCl3 (10 mL) at 70° C. for 3 h. MM-I-05 wasobtained as a white solid (5.1 g, 93%): mp 71-73° C.; 1H NMR (300 MHz,CDCl3) δ 9.30 (s, 1H; H-8), 8.56 (d, J=8.9 Hz, 1H; H-6), 8.46 (s, 1H;H-3), 7.89 (dd, J=8.9, 1.5 Hz, 1H; H-1), 4.54 (q, J=7.1 Hz, 2H; H-20),1.50 (t, J=7.1 Hz, 3H; H-19); 13C NMR (75 MHz, CDCl3) δ 163.96 (s),151.35 (s), 148.54 (s), 143.47 (s), 133.53 (q), 127.84 (s), 127.60 (q),126.92 (s), 124.69 (s), 124.01 (q), 118.86 (s), 62.45 (s), 14.21 (s);HRMS m/z calculated for C13H9NO2F3Cl304.0347 found 304.0353.

2-Methoxy-d3-5-nitropyridine [DK-II-44-1]. To a mixture of potassiumt-butoxide (13.3 g, 11.8 mmol) and methanol-d4 (50 mL) was slowly added2-chloro-5-nitropyridine (15.0 g, 94.6 mmol). The exothermic reactionwarmed to 50° C. and then was refluxed at 65° C. for 2 h to complete thereaction. The reaction mixture was then cooled to 20-25° C. and pouredinto water (750 mL). After stirring the mixture for 1 h the solidproduct was filtered and washed twice with water (25 mL×2). The solidwas dried to afford the product as a light yellow powder DK-II-44-1(13.0 g, 87%). ¹H NMR (300 MHz, DMSO) δ 9.08 (s, 1H), 8.47 (d, J=9.1 Hz,1H), 7.03 (d, J=9.1 Hz, 1H); ¹³C NMR (75 MHz, DMSO) δ 167.45, 145.02,139.99, 135.01, 111.67; HRMS m/z calculated for C6H4D3N203 (M+H)⁺158.0642 found 158.20.

5-Amino-2-methoxy-d3-pyridine [DK-II-45-1]. A mixture of2-methoxy-d3-5-ntiropyridine DK-II-44-1 (13.0 g, 82.7 mmol), iron powder(15.9 g, 284.7 mmol), water (5 mL) and ethanol (50 mL) was heated toreflux (78° C.). Once at reflux, concentrated hydrochloric acid (1 mL,83.3 mmol) was added dropwise. The reaction mixture was then refluxedfor 4 h to complete the reaction. Upon cooling to 20-25° C., the mixturewas filtered to remove the iron and the solids were washed 3 times withethanol (25 mL×3). Sodium bicarbonate (5.0 g) was added to the filtrateand the ethanol was removed by evaporation on a rotovap. Water (50 mL)and dichloromethane (50 mL) were added to dissolve the residue. Thelayers were separated and the aqueous layer was extract twice withdichloromethane (50 mL×2). The combined organic layers were dried overmagnesium sulfate. The solvents were then removed by evaporation on arotovap and the product was obtained as a clear orange-red oilDK-II-45-1 (10.0 g, 95.1%): ¹H NMR (300 MHz, DMSO) δ 7.57 (d, J=2.7 Hz,1H), 7.05 (dd, J=8.6, 2.9 Hz, 1H), 6.56 (d, J=8.7 Hz, 1H), 4.74 (s, 2H);¹³C NMR (75 MHz, DMSO) δ 156.29, 139.77, 131.63, 126.85, 110.42; HRMSm/z calculated for C6H6D3N2O (M+H)⁺ 128.0901 found 128.15.

4-Methoxy-d3-phenylhydrazine [DK-I-29-2]. A mixture ofN-(4-methoxy-d3-phenyl)acetamide DK-I-6-1 (30 g, 178.4 mmol),concentrated hydrochloric acid (72 mL), and water (72 mL) was heated toand held at 90° C. for 2 h to hydrolyze the amide functionality. Thereaction mixture was then cooled to 0 to 5° C. and a solution of sodiumnitrite (12.9 g, 187.7 mmol) and water (25 mL) was slowly addeddrop-wise to the reaction mixture. Upon completion of the addition, thereaction mixture was stirred for an additional 15 min at 0 to 5° C. Thereaction mixture was then cooled to −25 to −20° C. and a solution of tin(II) chloride (74.4 g, 392.4 mmol) and concentrated hydrochloric acid(150 mL) was added drop-wise to the reaction mixture over 30 min. Uponcompletion of the addition, the reaction mixture was stirred for anadditional 4 h at −25 to −20° C. The reaction mixture was then dilutedwith diethyl ether (300 mL) and the solids were filtered and washedthree times with diethyl ether (100 mL×3). The tin adduct of the productwas then dissolved in a mixture of sodium hydroxide (60 g), water (250mL) and dichloromethane (250 mL). After stirring for 2 h at 0 to 5° C.,the solids completely dissolved. The layers were separated and theaqueous layer was extracted three times with dichloromethane (100 mL×3).The combined organic layers were then dried over magnesium sulfate. Thesolvents were then removed by evaporation on a rotovap and the productresidue was slurried with hexanes (50 mL). The solid product was thenfiltered and washed twice with hexanes (50 mL×2). The solid was dried toafford the product as a pale orange crystalline solid DK-I-29-2 (16.6 g,66%): ¹H NMR (300 MHz, MeOD) δ 6.91-6.85 (m, 2H), 6.85-6.78 (m, 2H),4.88 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 152.05, 147.27, 114.60, 113.38;HRMS m/z calculated for C7H8D3N2O (M+H)⁺ 142.1057 found 142.25.

3-Methoxy-d3-phenylhydrazine [DK-I-26-3]. A mixture ofN-(3-methoxy-d3-phenyl)acetamide DK-I-8-1 (25 g, 148.6 mmol),concentrated hydrochloric acid (60 mL), and water (60 mL) was heated toand held at 90° C. for 2 h to hydrolyze the amide functionality. Thereaction mixture was then cooled to 0 to 5° C. and a solution of sodiumnitrite (10.8 g, 156.1 mmol) and water (21 mL) was slowly addeddrop-wise to the reaction mixture. Upon completion of the addition, thereaction mixture was stirred for an additional 15 min at 0 to 5° C. Thereaction mixture was then cooled to −25 to −20° C. and a solution of tin(II) chloride (62.0 g, 327.0 mmol) and concentrated hydrochloric acid(125 mL) was added drop-wise to the reaction mixture over 30 min. Uponcompletion of the addition, the reaction mixture was stirred for anadditional 2 h at −25 to −20° C. The reaction mixture was then dilutedwith diethyl ether (250 mL) and the solids were filtered and washedthree times with diethyl ether (100 mL×3). The tin adduct of the productwas then dissolved in a mixture of sodium hydroxide (20 g), water (100mL) and dichloromethane (100 mL). After stirring for 1 h at 0 to 5° C.,the solids completely dissolved. The layers were separated and theaqueous layer was extracted three times with dichloromethane (50 mL×3).The combined organic layers were then dried over magnesium sulfate. Thesolvents were then removed by evaporation on a rotovap to afford theproduct as an orange-red oil DK-I-26-3 (5.4 g, 26%): ¹H NMR (300 MHz,DMSO) δ 6.98 (t, J=8.0 Hz, 1H), 6.65 (s, 1H), 6.51-6.27 (m, 2H), 6.16(d, J=7.9 Hz, 1H), 3.91 (s, 2H); ¹³C NMR (75 MHz, DMSO) δ 160.68,154.54, 129.69, 104.96, 102.72, 97.52; HRMS m/z calculated for C7H8D3N20(M+H)⁺ 142.1057 found 142.15.

2-Methoxy-d3-phenylhydrazine [DK-I-43-3]. A mixture ofN-(2-methoxy-d3-phenyl)acetamide DK-I-31-1 (25 g, 148.6 mmol),concentrated hydrochloric acid (60 mL), and water (60 mL) was heated toand held at 90° C. for 2 h to hydrolyze the amide functionality. Thereaction mixture was then cooled to 0 to 5° C. and a solution of sodiumnitrite (10.7 g, 156.1 mmol) and water (21 mL) was slowly addeddrop-wise to the reaction mixture. Upon completion of the addition, thereaction mixture was stirred for an additional 15 min at 0 to 5° C. Thereaction mixture was then cooled to −25 to −20° C. and a solution of tin(II) chloride (62.0 g, 327.0 mmol) and concentrated hydrochloric acid(125 mL) was added drop-wise to the reaction mixture over 30 min. Uponcompletion of the addition, the reaction mixture was stirred for anadditional 2 h at −25 to −20° C. The reaction mixture was then dilutedwith diethyl ether (300 mL) and the solids were filtered and washedthree times with diethyl ether (100 mL×3). The tin adduct of the productwas then dissolved in a mixture of sodium hydroxide (20 g), water (100mL) and dichloromethane (100 mL). After stirring for 1 h at 0 to 5° C.,the solids completely dissolved. The layers were separated and theaqueous layer was extracted three times with dichloromethane (100 mL×3).The combined organic layers were then dried over magnesium sulfate. Thesolvents were then removed by evaporation on a rotovap and the productresidue was slurried with hexanes (50 mL). The solid product was thenfiltered and washed twice with hexanes (25 mL×2). The solid was dried toafford the product as a pale pink solid DK-I-43-3 (12.5 g, 60%): ¹H NMR(300 MHz, DMSO) δ 7.01 (dd, J=7.8, 1.3 Hz, 1H), 6.92-6.71 (m, 2H), 6.61(td, J=7.7, 1.4 Hz, 1H), 5.92 (s, 1H), 3.92 (s, 2H); ¹³C NMR (75 MHz,DMSO) δ 146.33, 141.94, 121.26, 117.23, 111.20, 110.07; HRMS m/zcalculated for C7H8D3N2O (M+H)⁺ 142.1057 found 142.30.

5-Hydrazinyl-2-methoxypyridine [DK-I-82-3]. A mixture of5-amino-2-methoxypyridine (10 g, 80.6 mmol), concentrated hydrochloricacid (24 mL), and water (24 mL) was cooled to 0 to 5° C. and a solutionof sodium nitrite (5.8 g, 84.6 mmol) and water (12 mL) was slowly addeddrop-wise to the reaction mixture. Upon completion of the addition, thereaction mixture was stirred for an additional 15 min at 0 to 5° C. Thereaction mixture was then cooled to −25 to −20° C. and a solution of tin(II) chloride (33.6 g, 177.2 mmol) and concentrated hydrochloric acid(70 mL) was added drop-wise to the reaction mixture over 30 min. Uponcompletion of the addition, the reaction mixture was stirred for anadditional 2 h at −25 to −20° C. The reaction mixture was then dilutedwith dichloromethane (100 mL). A solution of potassium hydroxide (100g), water (200 mL) was added dropwise to the reaction mixture at 0 to 5°C. over 30 min. After stirring for 1 h at 0 to 5° C., the solidscompletely dissolved. The layers were separated and the aqueous layerwas extracted four times with dichloromethane (50 mL×4). The combinedorganic layers were then dried over magnesium sulfate. The solvents werethen removed by evaporation on a rotovap and the product residue wasslurried with hexanes (20 mL). The slurry was placed in a freezer at−20° C. for 24 h to fully precipitate the product. The solid product wasthen filtered and washed twice with hexanes (10 mL×2). The solid wasdried to afford the product as a pale yellow-brown solid DK-I-82-3 (7.8g, 70%): ¹H NMR (300 MHz, DMSO) δ 7.70 (s, 1H), 7.20 (d, J=8.7 Hz, 1H),6.61 (d, J=8.8 Hz, 1H), 6.41 (s, 1H), 3.97 (s, 2H), 3.73 (s, 3H); ¹³CNMR (75 MHz, DMSO) δ 156.98, 144.09, 129.85, 125.25, 110.22, 53.19; HRMSm/z calculated for C6H10N3O (M+H)⁺ 140.0824 found 140.25.

5-Hydrazinyl-2-methoxy-d3-pyridine [DK-II-56-1]. A mixture of5-amino-2-methoxy-d3-pyridine DK-II-45-1 (10 g, 78.6 mmol), concentratedhydrochloric acid (24 mL), and water (24 mL) was cooled to 0 to 5° C.and a solution of sodium nitrite (5.7 g, 82.5 mmol) and water (12 mL)was slowly added drop-wise to the reaction mixture. Upon completion ofthe addition, the reaction mixture was stirred for an additional 15 minat 0 to 5° C. The reaction mixture was then cooled to −25 to −20° C. anda solution of tin (II) chloride (32.8 g, 173.0 mmol) and concentratedhydrochloric acid (70 mL) was added drop-wise to the reaction mixtureover 30 min. Upon completion of the addition, the reaction mixture wasstirred for an additional 2 h at −25 to −20° C. The reaction mixture wasthen diluted with dichloromethane (100 mL). A solution of potassiumhydroxide (100 g), water (200 mL) was added dropwise to the reactionmixture at 0 to 5° C. over 30 min. After stirring for 1 h at 0 to 5° C.,the solids completely dissolved. The layers were separated and theaqueous layer was extracted four times with dichloromethane (50 mL×4).The combined organic layers were then dried over magnesium sulfate. Thesolvents were then removed by evaporation on a rotovap and the productresidue was slurried with hexanes (20 mL). The slurry was placed in afreezer at −20° C. for 24 h to fully precipitate the product. The solidproduct was then filtered and washed twice with hexanes (10 mL×2). Thesolid was dried to afford the product as a pale yellow-brown solidDK-II-56-1 (6.4 g, 57%): ¹H NMR (300 MHz, DMSO) δ 7.70 (d, J=2.8 Hz,1H), 7.31-7.19 (m, 1H), 6.61 (d, J=8.8 Hz, 1H), 6.42 (s, 1H), 4.00 (s,2H); ¹³C NMR (75 MHz, DMSO) δ 157.01, 144.07, 129.87, 125.27, 110.22;HRMS m/z calculated for C6H7D3O (M+H)⁺ 143.1010 found 143.25.

7-Methoxy-2-(4-methoxyphenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[Comp 6]. A mixture of ethyl-4-chloro-7-methoxyquinoline-3-carboxylateDK-I-40-1 (4 g, 15.1 mmol), 4-methoxyphenylhydrazine hydrochloride (3.15g, 18.1 mmol), triethylamine (3.66 g, 36.1 mmol) and xylenes (32 mL) washeated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (32mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder Comp 6 (2.0 g, 41%): ¹H NMR(300 MHz, DMSO) δ 12.59 (s, 1H), 8.65 (s, 1H), 8.10 (t, J=8.7 Hz, 3H),7.34-7.12 (m, 2H), 7.01 (d, J=9.1 Hz, 2H), 3.87 (s, 3H), 3.78 (s, 3H);¹³C NMR (75 MHz, DMSO) δ 161.45, 160.85, 156.22, 143.11, 139.33, 137.42,134.10, 124.05, 120.68, 115.77, 114.25, 112.68, 106.87, 102.26, 55.98,55.68; HRMS m/z calculated for C18H16N3O3 (M+H)⁺ 322.1191 found 322.25.

7-Methoxy-d3-2-(4-methoxyphenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[RV-I-029]. A mixture ofethyl-4-chloro-7-methoxy-d3-quinoline-3-carboxylate DK-I-57-1 (2 g, 7.4mmol), 4-methoxyphenylhydrazine hydrochloride (1.56 g, 8.9 mmol),triethylamine (1.81 g, 17.6 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder RV-I-029 (1.0 g, 41%): ¹H NMR (300 MHz, DMSO)δ 12.59 (s, 1H), 8.65 (s, 1H), 8.10 (t, J=8.7 Hz, 3H), 7.17 (d, J=2.0Hz, 2H), 7.01 (d, J=8.9 Hz, 2H), 3.78 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ161.45, 160.86, 156.22, 143.11, 139.32, 137.42, 134.11, 124.05, 120.68,115.76, 114.25, 112.66, 106.87, 102.25, 55.68; HRMS m/z calculated forC18H13D3N3O3 (M+H)⁺ 325.1377 found 325.30.

7-Methoxy-2-(4-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-56-1]. A mixture ofethyl-4-chloro-7-methoxyquinoline-3-carboxylate DK-I-40-1 (2 g, 7.4mmol), 4-methoxy-d3-phenylhydrazine DK-I-29-2 (1.25 g, 8.9 mmol),triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-I-56-1 (1.5 g, 62.5%): ¹H NMR (300 MHz,DMSO) δ 12.60 (s, 1H), 8.66 (s, 1H), 8.10 (t, J=9.7 Hz, 3H), 7.18 (s,2H), 7.01 (d, J=8.4 Hz, 2H), 3.88 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ160.73, 160.43, 156.39, 143.09, 139.34, 137.43, 134.08, 124.08, 120.68,115.80, 114.24, 112.69, 106.87, 102.28, 56.00; HRMS m/z calculated forC18H13D3N3O3 (M+H)⁺ 325.1377 found 325.30.

7-Methoxy-d3-2-(4-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-60-3]. A mixture ofethyl-4-chloro-7-methoxy-d3-quinoline-3-carboxylate DK-I-57-1 (2 g, 7.4mmol), 4-methoxy-d3-phenylhydrazine DK-I-29-2 (1.26 g, 8.9 mmol),triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-I-60-3 (1.2 g, 50.0%): ¹H NMR (300 MHz,DMSO) δ 12.57 (s, 1H), 8.65 (s, 1H), 8.10 (t, J=9.0 Hz, 3H), 7.17 (d,J=5.7 Hz, 2H), 7.01 (d, J=9.0 Hz, 2H); ¹³C NMR (75 MHz, DMSO) δ 161.45,160.85, 156.21, 143.12, 139.37, 137.48, 134.10, 124.05, 120.69, 115.76,114.24, 112.68, 106.86, 102.30; HRMS m/z calculated for C18H10D6N3O3(M+H)⁺ 328.1569 found 328.15.

7-Methoxy-d3-2-(3-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-94-1]. A mixture ofethyl-4-chloro-7-methoxy-d3-quinoline-3-carboxylate DK-I-57-1 (2 g, 7.4mmol), 3-methoxy-d3-phenylhydrazine DK-I-26-3 (1.26 g, 8.9 mmol),triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-I-94-1 (1.5 g, 62.0%): ¹H NMR (300 MHz,DMSO) δ 12.62 (s, 1H), 8.66 (s, 1H), 8.13 (d, J=9.4 Hz, 1H), 7.93-7.73(m, 2H), 7.34 (t, J=8.2 Hz, 1H), 7.18 (d, J=6.7 Hz, 2H), 6.74 (d, J=8.2Hz, 1H); ¹³C NMR (75 MHz, DMSO) δ 162.13, 161.02, 159.97, 143.50,141.73, 139.59, 137.58, 129.94, 124.18, 115.84, 112.56, 111.30, 109.60,106.83, 104.81, 102.32; HRMS m/z calculated for C18H10D6N3O3 (M+H)⁺328.1569 found 328.25.

7-Methoxy-d3-2-(2-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-90-1]. A mixture ofethyl-4-chloro-7-methoxy-d3-quinoline-3-carboxylate DK-I-57-1 (2 g, 7.4mmol), 2-methoxy-d3-phenylhydrazine DK-I-43-3 (1.26 g, 8.9 mmol),triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-I-90-1 (1.8 g, 75.0%): ¹H NMR (300 MHz,DMSO) δ 12.47 (s, 1H), 8.57 (s, 1H), 7.99 (d, J=8.7 Hz, 1H), 7.51-7.24(m, 2H), 7.22-6.93 (m, 4H); ¹³C NMR (75 MHz, DMSO) δ 162.19, 160.63,155.66, 142.97, 139.04, 137.26, 129.88, 129.67, 128.51, 123.87, 120.65,115.49, 112.96, 112.91, 105.83, 102.15; HRMS m/z calculated forC18H10D6N3O3 (M+H)⁺ 328.1569 found 328.30.

7-Methoxy-2-(2-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-88-1]. A mixture ofethyl-4-chloro-7-methoxy-quinoline-3-carboxylate DK-I-40-1 (2 g, 7.5mmol), 2-methoxy-d3-phenylhydrazine DK-I-43-3 (1.28 g, 9.0 mmol),triethylamine (0.91 g, 9.0 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-I-88-1 (1.6 g, 65.6%): ¹H NMR (300 MHz,DMSO) δ 12.46 (d, J=4.9 Hz, 1H), 8.57 (d, J=5.8 Hz, 1H), 7.99 (d, J=8.7Hz, 1H), 7.40 (t, J=7.8 Hz, 1H), 7.31 (d, J=7.6 Hz, 1H), 7.15 (dd,J=9.7, 6.0 Hz, 3H), 7.03 (t, J=7.5 Hz, 1H), 3.87 (s, 3H); ¹³C NMR (75MHz, DMSO) δ 184.22, 162.19, 160.62, 155.66, 142.95, 139.04, 137.26,129.88, 129.66, 128.51, 123.88, 120.65, 115.49, 112.97, 112.94, 105.83,102.15, 55.94; HRMS m/z calculated for C18H13D3N3O3 (M+H)⁺ 325.1377found 325.25.

8-Chloro-2-(4-methoxyphenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[Comp 11]. A mixture of ethyl-4,6-dichloro-quinoline-3-carboxylateDK-I-35-1 (2 g, 7.4 mmol), 4-methoxyphenylhydrazine hydrochloride (1.55g, 8.9 mmol), triethylamine (1.80 g, 17.8 mmol) and xylenes (16 mL) washeated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (16mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder Comp 11 (1.7 g, 71.0%): ¹H NMR(300 MHz, DMSO) δ 12.95 (d, J=5.6 Hz, 1H), 8.72 (d, J=6.2 Hz, 1H), 8.14(d, J=2.1 Hz, 1H), 8.08 (d, J=9.0 Hz, 2H), 7.70 (dt, J=8.9, 5.5 Hz, 2H),7.02 (d, J=9.1 Hz, 2H), 3.79 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 161.37,156.48, 141.97, 139.79, 134.59, 133.87, 131.04, 130.46, 122.08, 121.50,120.89, 120.48, 114.29, 106.86, 55.71; HRMS m/z calculated forC17H13ClN3O2 (M+H)⁺ 326.0696 found 326.25.

8-Chloro-2-(4-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-93-1]. A mixture of ethyl-4,6-dichloro-quinoline-3-carboxylateDK-I-35-1 (2 g, 7.4 mmol), 4-methoxy-d3-phenylhydrazine DK-I-29-2 (1.25g, 8.9 mmol), triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL) washeated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (16mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solids was driedto afford the product as a yellow powder DK-I-93-1 (1.3 g, 53.6%): ¹HNMR (300 MHz, DMSO) δ 12.89 (s, 1H), 8.74 (s, 1H), 8.24-7.89 (m, 3H),7.86-7.56 (m, 2H), 7.02 (d, J=8.9 Hz, 2H); ¹³C NMR (75 MHz, DMSO) δ161.38, 156.49, 141.98, 139.86, 134.59, 133.84, 131.05, 130.49, 122.09,121.52, 120.91, 120.49, 114.29, 106.88; HRMS m/z calculated forC17H10D3ClN3O2 (M+H)⁺ 329.0882 found 329.15.

8-Chloro-2-(3-methoxyphenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[LAU 159]. A mixture of ethyl-4,6-dichloro-quinoline-3-carboxylateDK-I-35-1 (2 g, 7.4 mmol), 3-methoxyphenylhydrazine hydrochloride (1.55g, 8.9 mmol), triethylamine (1.80 g, 17.8 mmol) and xylenes (16 mL) washeated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (16mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder LAU 159 (0.7 g, 30.0%): ¹H NMR(300 MHz, DMSO) δ 12.85 (s, 1H), 8.69 (s, 1H), 8.15 (d, J=1.9 Hz, 1H),7.83 (d, J=8.7 Hz, 2H), 7.70 (dt, J=9.0, 5.4 Hz, 2H), 7.34 (t, J=8.1 Hz,1H), 6.83-6.65 (m, 1H), 3.81 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 161.99,159.98, 142.44, 141.52, 140.02, 134.81, 131.11, 130.62, 129.97, 122.17,121.62, 120.42, 111.47, 110.04, 106.80, 104.96, 55.59; HRMS m/zcalculated for C17H13ClN3O2 (M+H)⁺ 326.0696 found 326.20.

8-Chloro-2-(3-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-59-1]. A mixture of ethyl-4,6-dichloro-quinoline-3-carboxylateDK-I-35-1 (2 g, 7.4 mmol), 3-methoxy-d3-phenylhydrazine hydrochlorideDK-I-26-2 (1.45 g, 8.1 mmol), triethylamine (1.87 g, 18.5 mmol) andxylenes (16 mL) was heated to reflux (138° C.) and held at reflux for 2h. The resulting yellow-orange slurry was cooled to 100° C. and dilutedwith ethanol (16 mL). The reaction mixture was then refluxed at 80° C.for 30 min and then cooled to 20-25° C. The solids were collected byfiltration and washed twice with a 1:1 mixture of ethanol (2.5 mL×2) andhexanes (2.5 mL×2) and then washed twice with hexanes (5 mL×2). Thesolid was dried to afford the product as a yellow powder DK-I-59-1 (2.0g, 87.0%): ¹H NMR (300 MHz, DMSO) δ 12.85 (s, 1H), 8.71 (s, 1H), 8.17(s, 1H), 8.00-7.49 (m, 4H), 7.35 (t, J=7.7 Hz, 1H), 6.77 (d, J=7.4 Hz,1H); ¹³C NMR (75 MHz, DMSO) δ 162.01, 160.01, 142.48, 141.54, 140.10,134.76, 131.15, 130.72, 130.01, 122.14, 121.68, 120.45, 111.42, 110.04,106.87, 104.95; HRMS m/z calculated for C17H10D3ClN3O2 (M+H)⁺ 329.0882found 329.10.

8-Chloro-2-(2-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-87-1]. A mixture of ethyl-4,6-dichloro-quinoline-3-carboxylateDK-I-35-1 (2 g, 7.4 mmol), 2-methoxy-d3-phenylhydrazine DK-I-43-3 (1.25g, 8.9 mmol), triethylamine (0.9 g, 8.9 mmol) and xylenes (16 mL) washeated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (16mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder DK-I-87-1 (1.0 g, 41.0%): ¹HNMR (300 MHz, DMSO) δ 12.74 (s, 1H), 8.66 (s, 1H), 8.03 (s, 1H), 7.69(p, J=9.0 Hz, 2H), 7.42 (t, J=7.8 Hz, 1H), 7.32 (d, J=7.6 Hz, 1H), 7.17(d, J=8.3 Hz, 1H), 7.05 (t, J=7.5 Hz, 1H); ¹³C NMR (75 MHz, DMSO) δ162.15, 155.64, 141.87, 139.59, 134.48, 130.83, 130.23, 129.91, 129.85,128.22, 121.91, 121.40, 120.76, 120.68, 113.00, 105.81; HRMS m/zcalculated for C17H10D3ClN3O2 (M+H)⁺ 329.0882 found 329.20.

7-Bromo-2-(4-methoxyphenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[LAU 463]. A mixture of ethyl-7-bromo-4-chloro-quinoline-3-carboxylateDK-I-52-1 (2 g, 6.3 mmol), 4-methoxyphenylhydrazine hydrochloride (1.33g, 7.6 mmol), triethylamine (1.54 g, 15.3 mmol) and xylenes (16 mL) washeated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (16mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder LAU 463 (1.4 g, 60.0%): ¹H NMR(300 MHz, DMSO) δ 12.75 (s, 1H), 8.74 (s, 1H), 8.09 (dd, J=17.7, 8.8 Hz,3H), 7.89 (d, J=1.6 Hz, 1H), 7.68 (dd, J=8.6, 1.6 Hz, 1H), 7.02 (d,J=9.1 Hz, 2H), 3.79 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 161.37, 156.47,142.38, 140.08, 136.98, 133.85, 129.65, 124.51, 122.95, 122.22, 120.87,118.22, 114.31, 107.21, 55.71; HRMS m/z calculated for C17H13BrN3O2(M+H)⁺ 370.0191 found 370.15.

7-Bromo-2-(4-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-58-1]. A mixture of ethyl-7-bromo-4-chloro-quinoline-3-carboxylateDK-I-52-1 (2 g, 6.3 mmol), 4-methoxy-d3-phenylhydrazine DK-I-29-2 (1.08g, 7.6 mmol), triethylamine (0.77 g, 7.6 mmol) and xylenes (16 mL) washeated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (16mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder DK-I-58-1 (1.0 g, 42.0%): ¹HNMR (300 MHz, DMSO) δ 12.75 (s, 1H), 8.74 (d, J=4.9 Hz, 1H), 8.09 (dd,J=17.8, 8.7 Hz, 3H), 7.88 (s, 1H), 7.69 (d, J=8.4 Hz, 1H), 7.01 (d,J=8.8 Hz, 2H); ¹³C NMR (75 MHz, DMSO) δ 161.35, 156.49, 141.88, 140.06,136.95, 133.83, 129.65, 124.51, 122.93, 122.18, 120.86, 118.22, 114.24,107.22; HRMS m/z calculated for C17H10D3BrN3O2 (M+H)⁺ 373.0377 found373.05.

7-Bromo-2-(3-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-92-1]. A mixture of ethyl-7-bromo-4-chloro-quinoline-3-carboxylateDK-I-52-1 (1.5 g, 4.8 mmol), 3-methoxy-d3-phenylhydrazine DK-I-26-3(0.81 g, 5.7 mmol), triethylamine (0.58 g, 5.7 mmol) and xylenes (12 mL)was heated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (12mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder DK-I-92-1 (0.7 g, 40.0%): ¹HNMR (300 MHz, DMSO) δ 12.78 (s, 1H), 8.76 (s, 1H), 8.15 (d, J=8.5 Hz,1H), 7.95-7.76 (m, 3H), 7.71 (d, J=8.6 Hz, 1H), 7.35 (t, J=8.2 Hz, 1H),6.76 (d, J=8.3 Hz, 1H); ¹³C NMR (75 MHz, DMSO) δ 162.00, 160.00, 142.81,141.51, 140.33, 137.11, 130.03, 129.75, 124.65, 123.22, 122.26, 118.16,111.39, 109.98, 107.20, 104.92; HRMS m/z calculated for C17H10D3BrN3O2(M+H)⁺ 373.0377 found 373.10.

7-Bromo-2-(2-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-89-1]. A mixture of ethyl-7-bromo-4-chloro-quinoline-3-carboxylateDK-I-52-1 (2 g, 6.3 mmol), 2-methoxy-d3-phenylhydrazine DK-I-43-3 (1.08g, 7.6 mmol), triethylamine (0.77 g, 7.6 mmol) and xylenes (16 mL) washeated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (16mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder DK-I-89-1 (1.2 g, 50.6%): ¹HNMR (300 MHz, DMSO) δ 12.60 (s, 1H), 8.64 (d, J=16.3 Hz, 1H), 8.00 (d,J=8.5 Hz, 1H), 7.86 (s, 1H), 7.64 (d, J=8.5 Hz, 1H), 7.41 (t, J=7.8 Hz,1H), 7.32 (d, J=7.7 Hz, 1H), 7.17 (d, J=8.3 Hz, 1H), 7.04 (t, J=7.5 Hz,1H); ¹³C NMR (75 MHz, DMSO) δ 162.14, 158.75, 155.64, 142.23, 139.79,136.89, 129.84, 129.45, 128.23, 124.36, 122.64, 122.04, 120.69, 118.50,112.99, 106.18; HRMS m/z calculated for C17H10D3BrN3O2 (M+H)⁺ 373.0377found 373.10.

8-Methoxy-d3-2-(4-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-95-3]. A mixture ofethyl-4-chloro-6-methoxy-d3-quinoline-3-carboxylate DK-I-73-2 (2 g, 7.4mmol), 4-methoxy-d3-phenylhydrazine DK-I-29-2 (1.26 g, 8.9 mmol),triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-I-95-3 (0.9 g, 37.0%): ¹H NMR (300 MHz,DMSO) δ 12.77 (s, 1H), 8.64 (s, 1H), 8.11 (d, J=8.8 Hz, 2H), 7.68 (d,J=9.1 Hz, 1H), 7.57 (s, 1H), 7.28 (d, J=9.1 Hz, 1H), 7.02 (d, J=8.8 Hz,2H); ¹³C NMR (75 MHz, DMSO) δ 161.59, 157.98, 156.34, 143.00, 138.11,134.10, 130.13, 121.70, 120.92, 120.50, 119.95, 114.23, 105.70, 102.96;HRMS m/z calculated for C18H10D6N3O3 (M+H)⁺ 328.1569 found 328.25.

8-Methoxy-d3-2-(3-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-97-1]. A mixture ofethyl-4-chloro-6-methoxy-d3-quinoline-3-carboxylate DK-I-73-2 (2 g, 7.4mmol), 3-methoxy-d3-phenylhydrazine DK-I-26-3 (1.26 g, 8.9 mmol),triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-I-97-1 (1.8 g, 74.0%): ¹H NMR (300 MHz,DMSO) δ 12.80 (s, 1H), 8.65 (s, 1H), 7.99-7.80 (m, 2H), 7.67 (d, J=9.1Hz, 1H), 7.59 (s, 1H), 7.41-7.21 (m, 2H), 6.76 (d, J=8.2 Hz, 1H); ¹³CNMR (75 MHz, DMSO) δ 162.25, 159.98, 158.04, 143.42, 141.76, 138.36,130.26, 129.94, 121.73, 120.45, 120.10, 111.53, 109.64, 105.70, 105.11,103.16; HRMS m/z calculated for C18H10D6N3O3 (M+H)⁺ 328.1569 found328.30.

8-Methoxy-d3-2-(2-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-98-1]. A mixture ofethyl-4-chloro-6-methoxy-d3-quinoline-3-carboxylate DK-I-73-2 (2 g, 7.4mmol), 2-methoxy-d3-phenylhydrazine DK-I-43-3 (1.26 g, 8.9 mmol),triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-I-98-1 (0.5 g, 20.0%): ¹H NMR (300 MHz,DMSO) δ 12.65 (s, 1H), 8.57 (s, 1H), 7.65 (d, J=9.1 Hz, 1H), 7.54-7.28(m, 3H), 7.28-7.21 (m, 1H), 7.16 (d, J=8.3 Hz, 1H), 7.05 (t, J=7.5 Hz,1H); ¹³C NMR (75 MHz, DMSO) δ 162.38, 157.83, 155.76, 142.92, 137.98,130.05, 129.94, 129.83, 128.54, 121.50, 120.75, 120.65, 119.64, 112.91,104.61, 102.88; HRMS m/z calculated for C18H10D6N3O3 (M+H)⁺ 328.1569found 328.30.

7-Methoxy-2-(6-methoxypyridin-3-yl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-II-13-1]. A mixture ofethyl-4-chloro-7-methoxy-quinoline-3-carboxylate DK-I-40-1 (2 g, 7.5mmol), 5-hydrazinyl-2-methoxypyridine DK-I-82-3 (1.26 g, 9.0 mmol),triethylamine (0.91 g, 9.0 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-II-13-1 (1.2 g, 49.0%): ¹H NMR (300 MHz,DMSO) δ 12.65 (s, 1H), 8.92 (d, J=2.4 Hz, 1H), 8.68 (s, 1H), 8.43 (dd,J=9.0, 2.6 Hz, 1H), 8.24-7.91 (m, 1H), 7.29-7.02 (m, 2H), 6.92 (d, J=9.0Hz, 1H), 3.88 (s, 6H); ¹³C NMR (75 MHz, DMSO) δ 161.74, 160.98, 160.43,143.86, 139.71, 137.45, 137.37, 131.88, 130.77, 124.13, 115.94, 112.59,110.56, 106.22, 102.30, 56.00, 53.71; HRMS m/z calculated for C17H15N4O3(M+H)⁺ 323.1144 found 323.25.

7-Methoxy-d3-2-(6-methoxypyridin-3-yl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-I-86-1]. A mixture ofethyl-4-chloro-7-methoxy-d3-quinoline-3-carboxylate DK-I-57-1 (2 g, 7.4mmol), 5-hydrazinyl-2-methoxypyridine DK-I-82-3 (1.24 g, 8.9 mmol),triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-I-86-1 (1.0 g, 41.0%): ¹H NMR (300 MHz,DMSO) δ 12.69 (s, 1H), 8.92 (s, 1H), 8.69 (s, 1H), 8.43 (d, J=9.0 Hz,1H), 8.12 (d, J=9.4 Hz, 1H), 7.18 (s, 2H), 6.92 (d, J=9.0 Hz, 1H), 3.88(s, 3H); ¹³C NMR (75 MHz, DMSO) δ 161.75, 161.01, 160.44, 143.88,139.73, 137.46, 137.39, 131.88, 130.79, 124.15, 115.96, 112.57, 110.58,106.22, 102.31, 53.72; HRMS m/z calculated for C17H12D3N4O3 (M+H)⁺326.1330 found 326.20.

7-Methoxy-2-(6-methoxy-d3-pyridin-3-yl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-II-60-1]. A mixture ofethyl-4-chloro-7-methoxy-quinoline-3-carboxylate DK-I-40-1 (2 g, 7.5mmol), 5-hydrazinyl-2-methoxy-d3-pyridine DK-II-56-1 (1.28 g, 9.0 mmol),triethylamine (0.91 g, 9.0 mmol) and xylenes (16 mL) was heated toreflux (138° C.) and held at reflux for 2 h. The resulting yellow-orangeslurry was cooled to 100° C. and diluted with ethanol (16 mL). Thereaction mixture was then refluxed at 80° C. for 30 min and then cooledto 20-25° C. The solids were collected by filtration and washed twicewith a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5 mL×2) and thenwashed twice with hexanes (5 mL×2). The solid was dried to afford theproduct as a yellow powder DK-II-60-1 (1.2 g, 49.0%): ¹H NMR (300 MHz,DMSO) δ 12.68 (s, 1H), 8.91 (d, J=2.1 Hz, 1H), 8.68 (s, 1H), 8.42 (dd,J=9.0, 2.4 Hz, 1H), 8.16-8.03 (m, 1H), 7.18 (d, J=5.9 Hz, 2H), 6.92 (d,J=9.0 Hz, 1H), 3.87 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 161.74, 160.98,160.44, 143.85, 139.69, 137.44, 137.39, 131.86, 130.77, 124.13, 115.94,112.58, 110.54, 106.22, 102.29, 56.00; HRMS m/z calculated forC17H12D3N4O3 (M+H)⁺ 326.1330 found 326.30.

8-Chloro-2-(6-methoxypyridin-3-yl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-II-18-1]. A mixture of ethyl-4,6-dichloro-7-methoxy-3-carboxylateDK-1-35-1 (2 g, 7.4 mmol), 5-hydrazinyl-2-methoxypyridine DK-I-82-3(1.24 g, 8.9 mmol), triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL)was heated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (16mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder DK-II-18-1 (1.0 g, 41.0%): ¹HNMR (300 MHz, DMSO) δ 12.96 (s, 1H), 8.92 (d, J=2.6 Hz, 1H), 8.77 (s,1H), 8.42 (dd, J=8.9, 2.6 Hz, 1H), 8.14 (s, 1H), 7.89-7.60 (m, 2H), 6.93(d, J=9.0 Hz, 1H), 3.89 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 161.62,160.64, 142.70, 140.12, 137.57, 134.58, 131.66, 131.15, 130.92, 130.64,122.11, 121.58, 120.38, 110.59, 106.25, 53.74; HRMS m/z calculated forC16H12ClN4O2 (M+H)⁺ 327.0649 found 327.25.

8-Chloro-2-(6-methoxy-d3-pyridin-3-yl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-II-59-1]. A mixture of ethyl-4,6-dichloro-quinoline-3-carboxylateDK-1-35-1 (2 g, 7.4 mmol), 5-hydrazinyl-2-methoxy-d3-pyridine DK-II-56-1(1.26 g, 8.9 mmol), triethylamine (0.90 g, 8.9 mmol) and xylenes (16 mL)was heated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (16mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder DK-II-59-1 (1.4 g, 57.0%): ¹HNMR (300 MHz, DMSO) δ 12.92 (s, 1H), 8.90 (d, J=1.7 Hz, 1H), 8.74 (d,J=9.1 Hz, 1H), 8.40 (dd, J=8.9, 2.4 Hz, 1H), 8.09 (s, 1H), 7.78-7.61 (m,2H), 6.90 (d, J=8.9 Hz, 1H); ¹³C NMR (75 MHz, DMSO) δ 161.60, 160.63,142.67, 140.06, 137.55, 134.55, 131.64, 131.13, 130.87, 130.60, 122.07,121.56, 120.37, 110.55, 106.26; HRMS m/z calculated for C16H9D3ClN4O2(M+H)⁺ 330.0835 found 330.25.

7-Bromo-2-(6-methoxypyridin-3-yl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-II-48-1]. A mixture of ethyl-7-bromo-4-chloroquinoline-3-carboxylateDK-I-52-1 (2 g, 6.3 mmol), 5-hydrazinyl-2-methoxypyridine DK-I-82-3(1.06 g, 7.6 mmol), triethylamine (0.77 g, 7.6 mmol) and xylenes (16 mL)was heated to reflux (138° C.) and held at reflux for 2 h. The resultingyellow-orange slurry was cooled to 100° C. and diluted with ethanol (16mL). The reaction mixture was then refluxed at 80° C. for 30 min andthen cooled to 20-25° C. The solids were collected by filtration andwashed twice with a 1:1 mixture of ethanol (2.5 mL×2) and hexanes (2.5mL×2) and then washed twice with hexanes (5 mL×2). The solid was driedto afford the product as a yellow powder DK-II-48-1 (1.6 g, 67.0%): ¹HNMR (300 MHz, DMSO) δ 12.81 (s, 1H), 10.29-10.27 (m, 1H), 8.89 (s, 1H),8.75 (s, 1H), 8.40 (dd, J=8.9, 2.2 Hz, 1H), 8.09 (d, J=8.5 Hz, 1H), 7.86(s, 1H), 7.68 (d, J=8.1 Hz, 1H), 6.91 (d, J=8.9 Hz, 1H), 3.88 (s, 3H);¹³C NMR (75 MHz, DMSO) δ 161.63, 160.61, 143.11, 140.38, 137.53, 136.95,131.65, 130.88, 129.74, 124.54, 123.15, 122.26, 118.12, 110.61, 106.58,53.74; HRMS m/z calculated for C16H12BrN4O2 (M+H)⁺ 371.0143 found371.20.

7-Bromo-2-(6-methoxy-d3-pyridin-3-yl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[DK-II-58-1]. A mixture of ethyl-7-bromo-4-chloroquinoline-3-carboxylateDK-I-52-1 (1.20 g, 3.8 mmol), 5-hydrazinyl-2-methoxy-d3-pyridineDK-II-56-1 (0.65 g, 4.6 mmol), triethylamine (0.46 g, 4.6 mmol) andxylenes (16 mL) was heated to reflux (138° C.) and held at reflux for 2h. The resulting yellow-orange slurry was cooled to 100° C. and dilutedwith ethanol (16 mL). The reaction mixture was then refluxed at 80° C.for 30 min and then cooled to 20-25° C. The solids were collected byfiltration and washed twice with a 1:1 mixture of ethanol (2.5 mL×2) andhexanes (2.5 mL×2) and then washed twice with hexanes (5 mL×2). Thesolid was dried to afford the product as a yellow powder DK-II-58-1 (0.5g, 35.0%): ¹H NMR (300 MHz, DMSO) δ 12.77 (s, 1H), 8.88 (d, J=2.3 Hz,1H), 8.75 (d, J=8.4 Hz, 1H), 8.39 (dd, J=8.9, 2.5 Hz, 1H), 8.07 (d,J=8.5 Hz, 1H), 7.83 (s, 1H), 7.65 (d, J=8.6 Hz, 1H), 6.90 (d, J=8.9 Hz,1H); ¹³C NMR (75 MHz, DMSO) δ 161.60, 160.60, 143.08, 140.33, 137.51,136.93, 131.63, 130.83, 129.70, 124.50, 123.11, 122.24, 118.10, 110.56,106.58; HRMS m/z calculated for C16H9D3BrN4O2 (M+H)⁺ 374.0329 found374.20.

7-Methoxy(d3)-2-(phenyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one [RV-I-37].A mixture of ethyl-4-chloro-7-methoxy-d3-quinoline-3-carboxylateDK-I-57-1 (0.01 mol, 0.324 g), phenylhydrazine hydrochloride (0.012 mol,0.172 g) and TEA (0.012 mol, 0.12 g) in 40 mL xylene was refluxed for 4hr, cooled to room temperature. The precipitated compound was collectedby filtration. The compound was recrystallized from methanol as a yellowcolored compound RV-I-37, yield 75%, 0.22 g: mp>260° C. dec. 1H NMR (500MHz, MeOD) 8.5 (s, 1H), 8.236(d, 1H, J=9.0 Hz), 8.102(d, 2H, J=9.0),7.492-7.236(m, 5H,); 13C (125 MHz, MeOD) 161.4, 160.8, 156.2, 143.1,139.3, 137.4, 137.1, 134.1, 124.0, 120.6, 115.8, 114.2, 112.6, 106.8,102.2, 78.5, 55.69; HRMS m/z calculated for C17H11D3N3O2 295.1274 found295.1272.

2-(4-Methoxyphenyl)-2H-pyrazolo[4,3-c][1,5]naphthyridin-3(5H)-one[RV-I-071]. A mixture of ethyl 4-chloro-1,5-naphthyridine-3-carboxylate(0.01 mol, 0.236 g), 4-methoxyphenylhydrazine hydrochloride (0.012 mol,0.153 g), triethylamine (0.012mol, 0.12 g) and xylenes (40 mL) washeated to reflux (138° C.) and held at reflux for 4 hours. The resultingyellow-orange slurry was cooled to room temperature and the solids werecollected by filtration. The solids washed twice with 20 ml water.Drying of the solid afforded the product as a yellow powder RV-I-071(0.268 g): ¹H NMR (300 MHz, DMSO) δ 12.9 (s, 1H), 8.79 (s, 1H), 8.77 (s,1H), 8.11-8.10(d, 2H, J=9.0), 8.08 (s, 1H), 7.70-7.68 (m, 1H),7.05-7.03(d, 2H, J=9.0), 3.80 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 161.49,160.85, 156.56, 148.79.11, 143.06, 139.81, 136.60, 133.97,132.95,127.93, 125.21, 120.91, 114.33, 109.42, 55.72; HRMS m/z calculated forC16H12N4O2 (M+H)⁺ 293.1039 found 293.1037.

8-Bromo-6-fluoro-2-(4-methoxyphenyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one[MM-I-03]. A mixture of 0.5 g (1.5 mmol) of ethyl6-bromo-4-chloro-8-fluoroquinoline-3-carboxylate MM-I-02, 0.31 g (1.8mmol) of (4-methoxyphenyl)hydrazine hydrochloride and Et3N (2 mL) wasplaced in a flask with xylene (8 mL) and heated for 4 h. The reactionwas cooled at rt and filtered. The solid was washed several times withhexane and water. Then, the solid was dissolved in a base solution 3 NNaOH and stirred for 15 min. The base solution was neutralized with 3 NHCl and filtrated. The solid was recrystallized using hot ethanol anddried in vacuo, affording a yellow solid MM-I-03 (0.28 g, 48%): mp333-334° C.; 1H NMR (300 MHz, DMSO) δ 8.51 (s, 1H; H-6), 8.09 (s, 1H;H-8), 8.03 (d, J=8.9 Hz, 2H; H-15 and H-19), 7.89 (d, J=10.5 Hz, 1H;H-2), 7.01 (d, J=9.0 Hz, 2H; H-16 and H-18), 3.78 (s, 3H; H-24); 13C NMR(75 MHz, DMSO) δ 161.11 (s), 156.65 (s), 140.93 (s), 139.49 (s), 133.57(s), 124.37 (s), 124.19 (s), 122.02 (s), 121.00 (s), 120.61 (s), 119.08(q, J=3.3, 1.9 Hz), 118.82 (s), 118.34 (s), 118.22 (s), 114.32 (s),107.83 (s), 55.73 (s); HRMS m/z calculated for C17H11N3O2FBr 388.0091found 388.0094.

2-(4-Methoxyphenyl)-7-(trifluoromethyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one[MM-I-06]. Treatment of ethyl4-chloro-7-(trifluoromethyl)quinoline-3-carboxylate MM-I-05 (0.5 g, 1.5mmol) with (4-methoxyphenyl)hydrazine hydrochloride (0.57 g, 1.8 mmol)and Et3N (2 mL) in 8 mL of xylene under reflux for 4 h afforded thecorresponding product. The reaction was cooled at rt and filtered. Thesolid was washed several times with hexane and water. An acid-basecrystallization was needed to remove the triethylamine salt, and itafforded yellow crystals MM-I-06 (0.51 g, 82%): mp 315-316° C.; 1H NMR(300 MHz, DMSO) δ 12.93 (s, 1H; H-7), 8.84 (s, 1H; H-8), 8.40 (d, J=8.4Hz, 1H; H-6), 8.08 (d, J=9.1 Hz, 2H; H-15 and H-19), 8.03 (s, 1H; H-3),7.83 (d, J=8.1 Hz, 1H; H-1), 7.03 (d, J=9.1 Hz, 2H; H-16 and H-18), 3.79(s, 3H; H-22); 13C NMR (75 MHz, DMSO) δ 161.38 (s), 156.60 (s), 141.99(s), 140.79 (s), 135.83 (s), 133.75 (s), 130.23 (s), 129.80 (s), 125.96(s), 124.05 (s), 122.66 (q), 122.35 (s), 122.12 (s), 121.00 (s), 117.21(q), 114.34 (s), 107.42 (s), 55.71 (s); HRMS m/z calculated forC18H12N3O2F3 360.0954 found 360.0943.

2-(4-Chlorophenyl)-7-(trifluoromethyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one[MM-I-08]. The reaction of 0.5 g (1.6 mmol) of ethyl4-chloro-7-(trifluoromethyl)quinoline-3-carboxylate MM-I-05 with(4-chlorophenyl)hydrazine hydrochloride (0.47 g, 3.2 mmol) and 2 mL ofEt₃N in 8 mL of xylene at reflux for overnight afforded the product. Therecrystallization of solid gave yellow crystals MM-I-08 (0.44 g, 75%):mp 346-347° C.; 1H NMR (300 MHz, DMSO) δ 12.98 (s, 1H; H-7), 8.84 (s,1H; H-8), 8.34 (d, J=8.3 Hz, 1H; H-1), 8.21 (d, J=8.9 Hz, 2H; H-15 andH-19), 7.98 (s, 1H; H-3), 7.80 (d, J=8.3 Hz, 1H; H-6), 7.47 (d, J=8.9Hz, 2H; H-18 and H-16); 13C NMR (75 MHz, DMSO) δ 161.92 (s), 142.71 (s),141.03 (s), 139.11 (s), 135.84 (s), 130.51 (s), 130.08 (s), 129.08 (s),128.45 (s), 125.88 (s), 124.11 (s), 122.72 (q), 122.27 (s), 121.92 (s),120.40 (s), 117.15 (q), 107.15 (s). HRMS m/z calculated forC17H9N30F3Cl364.0459 found 364.0453.

2-(4-Nitrophenyl)-7-(trifluoromethyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one[MM-I-09]. In a flask containing ethyl4-chloro-7-(trifluoromethyl)quinoline-3-carboxylate MM-I-05 (0.2 g, 0.66mmol), (4-nitrophenyl)hydrazine (0.25 g, 1.3 mmol) and xylene (8 mL),was added 2 mL of Et3N and the flask was immediately placed in oil bathpreviously heated at 150° C. After 4 h of heating, the solid wascollected by filtration and washed with hexane and water. The sameprocedure of acid-base crystallization was used affording reddish solidMM-I-09 (0.035 g, 15%): mp>350° C.; 1H NMR (300 MHz, DMSO) δ 13.09 (s,1H; H-7), 8.89 (s, 1H; H-8), 8.41 (d, J=9.1 Hz, 2H; H-16 and H-18), 8.35(d, J=8.4 Hz, 1H; H-6), 8.27 (d, J=9.1 Hz, 2H; H-15 and H-19), 7.95 (s,1H; H-3), 7.82 (d, J=8.2 Hz, 1H; H-1); HRMS m/z calculated forC17H9N4O3F3 375.0700 found 375.0695.

2-(4-(Trifluoromethoxy)phenyl)-7-(trifluoromethyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one[MM-I-10]. A mixture of ethyl4-chloro-7-(trifluoromethyl)quinoline-3-carboxylate MM-I-05 (0.3 g, 1mmol) with (4-(trifluoromethoxy)phenyl)hydrazine hydrochloride (0.48 g,2 mmol) and Et3N (2 mL) in 8 mL of xylene was heated at reflux forovernight. The solid was collected by filtration and washed with hexaneand water. The solid was dissolved in 3 N NaOH solution (10 mL) andprecipitated with 3 N HCl (11 mL) solution. Then, a recrystallizationusing 15 mL EtOH and 2 mL of water was used and afforded yellow crystalsMM-I-10 (0.25 g, 60%): mp 286-287° C.; 1H NMR (300 MHz, DMSO) δ 8.86 (s,1H; H-8), 8.36 (d, J=8.4 Hz, 1H; H-6), 8.29 (d, J=9.0 Hz, 2H; H-15 andH-19), 7.99 (s, 1H; H-3), 7.81 (d, J=8.3 Hz, 1H; H-1), 7.43 (d, J=8.8Hz, 2H; H-16 and H-18); 13C NMR (75 MHz, DMSO) δ 161.98 (s), 144.85 (s),142.85 (s), 141.22 (s), 139.27 (s), 135.96 (s), 130.54 (s), 130.11 (s),124.12 (s), 122.78 (s), 122.29 (q, J=2.8 Hz), 121.98 (s), 120.37 (s),117.25 (q, J=8.4, 4.7 Hz), 107.07 (s); HRMS m/z calculated forC18H9N3O2F6 414.0672 found 414.0674.

2-(4-Fluorophenyl)-7-(trifluoromethyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one[MM-I-11]. Treatment of ethyl4-chloro-7-(trifluoromethyl)quinoline-3-carboxylate MM-I-05 (0.2 g, 0.66mmol) with (4-fluorophenyl)hydrazine hydrochloride (0.22 g, 1.3 mmol)and Et3N (2 mL) in 8 mL of xylene under reflux for overnight affordedthe corresponding product. The yellow crystals MM-I-11 (0.15 g, 65%)were obtained by recrystallization with hot EtOH: mp 296-297° C.; 1H NMR(300 MHz, DMSO) δ 12.98 (s, 11H; H-7), 8.84 (s, 11H; H-8), 8.36 (d,J=8.3 Hz, 1H; H-6), 8.19 (dd, J=9.0, 5.1 Hz, 2H; H-15 and H-19), 8.00(s, 1H; H-3), 7.81 (d, J=8.4 Hz, 1H; H-1), 7.27 (t, J=8.9 Hz, 2H; H-16and H-18); 13C NMR (75 MHz, DMSO) δ 161.69 (s), 160.85 (s), 157.66 (s),142.41 (s), 140.94 (s), 136.74 (s), 135.78 (s), 130.21 (q), 124.09 (d),124.07 (s), 122.73 (q), 121.99 (s), 120.97 (d), 117.14 (q), 115.95 (s),115.65 (s), 107.21 (s); HRMS m/z calculated for C17H9N3OF4 348.0755found 348.0766.

2-(3-Methoxyphenyl)-7-(trifluoromethyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one[MM-I-12]. The reaction of 0.5 g (1.6 mmol) of ethyl4-chloro-7-(trifluoromethyhquinoline-3-carboxylate MM-I-05 with(3-methoxyphenyl)hydrazine hydrochloride (0.575 g, 4.1 mmol) and 2 mL ofEt₃N in 15 mL of xylene at reflux for overnight afforded the product.Recrystallization gave yellow crystals MM-I-12 (0.762 g, 46%): mp>350°C.; ¹H NMR (500 MHz, DMSO) δ 8.85 (s, 1H, H-8), 8.43 (d, J=8.3 Hz, 1H,H-6), 8.04 (s, 1H, H-15), 7.88-7.80 (m, 3H, H-1 H-3 and H-19), 7.37 (t,J=8.2 Hz, 1H, H-18), 6.79 (dd, J=8.2, 2.4 Hz, 1H, H-17), 3.82 (s, 3H,H-22).

8-Bromo-2-(4-chlorophenyl)-6-fluoro-2H-pyrazolo[4,3-c]quinolin-3(5H)-one[MM-I-13]. A mixture of ethyl6-bromo-4-chloro-8-fluoroquinoline-3-carboxylate MM-I-02 (0.2 g, 0.64mmol) with (4-chlorophenyl)hydrazine hydrochloride (0.18 g, 1.2 mmol)and Et₃N (2 mL) in 8 mL of xylene was heated at reflux for overnight.The solid was collected by filtration and washed with hexane and water.The solid was dissolved in 10 mL of DMSO. The solution was poured in 30mL of H₂O, and filtered in order to remove the triethylamine salt. Then,a recrystallization using 15 mL EtOH and 2 mL of water was used andafforded yellow crystals MM-I-13 (0.17 g, 70%): ¹H NMR (300 MHz, DMSO) δ8.54 (s, 1H, H-8), 8.22 (d, J=8.9 Hz, 2H, H-15 and H-19), 8.11 (s, 1H,H-6), 7.92 (dd, J=10.6, 1.8 Hz, 1H, H-2), 7.50 (d, J=8.9 Hz, 2H, H-16and H-18).

8-Bromo-6-fluoro-2-(4-fluorophenyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one[MM-I-18]. Treatment of ethyl6-bromo-4-chloro-8-fluoroquinoline-3-carboxylate MM-I-02 (0.2 g, 0.64mmol) with (4-fluorophenyl)hydrazine hydrochloride (0.22 g, 1.3 mmol)and Et₃N (2 mL) in 8 mL of xylene under reflux overnight afforded thecorresponding product. The yellow crystals MM-I-18 (0.120 g, 50%) wereobtained by recrystallization with hot EtOH: ¹H NMR (300 MHz, DMSO) δ13.02 (s, 1H, H-7), 8.55 (s, 1H, H-8), 8.25-8.15 (m, 2H, H-15 and H-19),8.12 (s, 1H, H-6), 7.93 (dd, J=10.6, 1.9 Hz, 1H, H-2), 7.29 (t, J=8.9Hz, 2H, H-16 and H-18).

7-Methoxy-2-(4-(trifluoromethoxy)phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[CW-02-082]. To a clean and dry flask ethyl4-chloro-7-methoxyquinoline-3-carboxylate DK-I-40-1 (531 mg, 2 mmol, 1EQ), (4-(trifluoromethoxy)phenyl)hydrazine hydrochloride (686 mg, 3mmol, 1.5 EQ), xylene (10 mL), and TEA (607 mg, 6 mmol, 3 EQ) werecharged. The mixture was immediately transferred to pre-heated oil bath(150° C.) and heated overnight at which point it was cooled to 0° C. viaice/water bath and hexanes (20 mL) were added in one portion. The yellowsolid was filtered and dried (1.35 g product+TEA*HCl). The mixture waspurified via general purification method A and B. The solid was driedovernight under high vacuum obtaining the pure product in 75% yield (563mg) as a yellow powder CW-02-082: ¹H NMR (300 MHz, DMSO) δ 12.71 (s,1H), 8.71 (s, 1H), 8.33 (s, 2H), 8.12 (s, 1H), 7.45 (s, 2H), 7.18 (s,2H), 3.88 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ 162.18, 161.10, 144.50,144.48, 143.97, 139.93, 139.60, 137.61, 124.15, 121.98, 120.12, 115.95,112.52, 106.45, 102.37, 56.00; HRMS (ESI) (M+H),Calcd. for C18H13F3N3O3376.0909; Found 376.0914.

7-Methoxy-2-(4-methoxyphenyl)-6-methyl-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[CW-02-073]. To a clean and dry flask ethyl4-chloro-7-methoxy-8-methylquinoline-3-carboxylate (560 mg, 2 mmol, 1EQ), (4-methoxyphenyl) hydrazine hydrochloride (524 mg, 3 mmol, 1.5 EQ),xylene (10 mL), and TEA (607 mg, 6 mmol, 3 EQ) were charged. The mixturewas immediately transferred to pre-heated oil bath (150° C.) and heatedovernight at which point it was cooled to 0° C. via ice/water bath andhexanes (20 mL) were added in one portion. The yellow solid was filteredand dried (1.15 g product+TEA*HCl). The mixture was purified via generalpurification method B. The solid was dried overnight under high vacuumobtaining the pure product in 70% yield (470 mg) as a yellow solid.¹HNMR (300 MHz, DMSO) δ 11.80 (s, 1H), 8.37 (s, 1H), 8.08 (dd, J=8.9, 5.1Hz, 3H), 7.29 (d, J=9.0 Hz, 1H), 7.01 (d, J=9.1 Hz, 2H), 3.92 (s, 3H),3.78 (s, 3H), 2.36 (s, 3H).¹³C NMR (75 MHz, DMSO) δ 161.39, 158.24,156.22, 143.58, 139.11, 135.37, 134.09, 121.18, 120.66, 114.33, 114.25,112.80, 111.11, 106.53, 56.65, 55.68, 10.03.HRMS (ESI) (M+H), Calcd. forC19H18N3O3 336.1348; Found 336.1240

7-Methoxy-6-methyl-2-phenyl-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[CW-02-078]. To a clean and dry flask ethyl4-chloro-7-methoxy-8-methylquinoline-3-carboxylate (560 mg, 2 mmol, 1EQ), phenylhydrazine hydrochloride (434 mg, 3 mmol, 1.5 EQ), xylene (10mL), and TEA (607 mg, 6 mmol, 3 EQ) were charged. The mixture wasimmediately transferred to pre-heated oil bath (150° C.) and heatedovernight at which point it was cooled to 0° C. via ice/water bath andhexanes (20 mL) were added in one portion. The yellow solid was filteredand dried (1.17 g product+TEA*HCl). The mixture was purified via generalpurification method A. The solid was dried overnight under high vacuumobtaining the pure product in 84% yield (513 mg) as yellow crystals.¹HNMR (500 MHz, DMSO) δ 11.86 (s, 1H), 8.42 (s, 1H), 8.22 (d, J=8.2 Hz,2H), 8.11 (d, J=8.8 Hz, 1H), 7.45 (t, J=7.9 Hz, 2H), 7.33 (d, J=8.9 Hz,1H), 7.17 (t, J=7.3 Hz, 1H), 3.95 (s, 3H), 2.39 (s, 3H). ¹³C NMR (126MHz, DMSO) δ 162.00, 158.41, 144.04, 140.60, 139.43, 135.49, 129.15,124.29, 121.32, 118.98, 114.42, 112.77, 111.22, 106.48, 56.71, 10.08.HRMS (ESI) (M+H), Calcd. for C18H16N3O2 306.1243; Found 306.1237

7-Methoxy-6-methyl-2-(4-(trifluoromethoxy)phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one[CW-02-079]. To a clean and dry flask ethyl4-chloro-7-methoxy-8-methylquinoline-3-carboxylate (560 mg, 2 mmol, 1EQ), (4-(trifluoromethoxy) phenyl) hydrazine (689 mg, 3 mmol, 1.5 EQ),xylene (10 mL), and TEA (607 mg, 6 mmol, 3 EQ) were charged. The mixturewas immediately transferred to pre-heated oil bath (150° C.) and heatedovernight at which point it was cooled to 0° C. via ice/water bath andhexanes (20 mL) were added in one portion. The yellow solid was filteredand dried (1.14 g product+TEA*HCl). The mixture was purified via generalpurification method B. The solid was dried overnight under high vacuumobtaining the pure product in 68% yield (530 mg) as a yellow powder. ¹HNMR (300 MHz, DMSO) δ 11.92 (d, J=5.8 Hz, 1H), 8.44 (d, J=6.3 Hz, 1H),8.33 (d, J=9.1 Hz, 2H), 8.09 (d, J=8.9 Hz, 1H), 7.45 (d, J=8.9 Hz, 2H),7.32 (d, J=9.0 Hz, 1H), 3.94 (s, 3H), 2.37 (s, 3H). ¹³C NMR (75 MHz,CDCl₃) δ 162.11, 158.52, 144.48, 144.44, 139.74, 139.57, 135.51, 122.35,122.00, 121.34, 120.10, 114.51, 112.64, 111.31, 106.11, 56.70, 10.05.HRMS (ESI) (M+H), Calcd. for C19H15F3N3O3 390.1066; Found 390.1068.

7-Methoxy-2-(4-methoxyphenyl)-2,5-dihydro-3H-pyrazolo[4,3-c][1,6]naphthyridin-3-one[CW-03-030]. To a clean and dry flask ethyl4-chloro-7-methoxy-1,6-naphthyridine-3-carboxylate (78 mg, 0.29 mmol, 1EQ), (4-methoxyphenyl)hydrazine hydrochloride (76 mg, 0.435 mmol, 1.5EQ), xylene (5 mL), and TEA (89 mg, 0.87 mmol, 3 EQ) were charged. Themixture was immediately transferred to pre-heated oil bath (150° C.) andheated overnight at which point it was cooled to 0° C. via ice/waterbath and hexanes (20 mL) were added in one portion. The yellow solid wasfiltered and dried (product+TEA*HCl). The mixture was purified via FCC(7% MeOH in DCM) and general purification method A. The solid was driedovernight under high vacuum obtaining the pure product in 47% yield (44mg) as orange crystals. ¹H NMR (300 MHz, DMSO) δ 12.60 (s, 1H), 9.06 (s,1H), 8.67 (s, 1H), 8.02 (d, J=9.1 Hz, 2H), 7.02 (d, J=9.1 Hz, 2H), 6.89(s, 1H), 3.96 (s, 3H), 3.79 (s, 3H). ¹³C NMR (75 MHz, DMSO) δ 164.59,161.30, 156.46, 144.09, 143.92, 141.36, 141.25, 133.63, 120.84, 114.32,110.13, 108.40, 96.94, 55.72, 54.49. HRMS (ESI) (M+H), Calcd. forC17H15N4O3 323.1144; Found 323.1138.

7-Chloro-2-(4-methoxyphenyl)-2,5-dihydro-3H-pyrazolo[4,3-c][1,6]naphthyridin-3-one[CW-03-033]. To a clean and dry flask ethyl4,7-dichloro-1,6-naphthyridine-3-carboxylate (120 mg, 0.443 mmol, 1 EQ),(4-methoxyphenyl)hydrazine hydrochloride (116 mg, 0.664 mmol, 1.5 EQ),xylene (5 mL), and TEA (135 mg, 1.33 mmol, 3 EQ) were charged. Themixture was immediately transferred to pre-heated oil bath (150° C.) andheated overnight at which point it was cooled to 0° C. via ice/waterbath and hexanes (15 mL) were added in one portion. The yellow solid wasfiltered and dried (product+TEA*HCl). The mixture was purified via FCC(7% MeOH in DCM) and general purification method B. The solid was driedovernight under high vacuum obtaining the pure product in 61% yield (88mg) as an orange solid. ¹H NMR (300 MHz, DMSO) δ 12.87 (s, 1H), 9.21 (s,1H), 8.81 (s, 1H), 8.03 (d, J=9.1 Hz, 2H), 7.61 (s, 1H), 7.03 (d, J=9.1Hz, 2H), 3.79 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 161.08, 156.70, 150.07,146.16, 143.21, 141.60, 140.52, 133.41, 121.00, 114.38, 114.10, 113.06,109.77, 55.73. HRMS (ESI) (M+H), Calcd. for C16H12ClN4O2 327.0649; Found327.0654.

Purification Method A: Crude compound was heated to reflux in ethanol(10 mL/g) and an aqueous ethanolic solution (50% H₂O, 50% ethanol) wasslowly added until all compound was completely dissolved at reflux. Oncedissolved, water was added dropwise at reflux until the solution becameslightly cloudy. The solution was cooled slowly to RT and in most casesmicrocrystals began to accumulate. The solution was further cooled to 0°C. via ice-bath, filtered, and washed with cold aqueous ethanolicsolution (50% H₂O, 50% ethanol) overnight. If necessary, compounds werefurther dried under high vacuum.

Purification Method B: Crude compound was dissolved in minimal DMSO andwater was added until compound was completely precipitated from the DMSOsolution. The product is filtered and washed with water. The amorphouspowder was ground with mortar and pestle and dried under high vacuum for24 hr's or until little trace of water is present in HNMR.

Example 2 Materials and Methods Animal Experiments

All animal experiments conducted at NTU were approved by theInstitutional Animal Care and Use Committee of College of Medicine,National Taiwan University. C57/B6 (8-10 weeks) mice were used for thePPI test and Wistar rats (8-9 weeks) were used in the migraine model.Animals were housed in an animal room with a 12-h light/12-h dark cycleand free access to food and water.

PPI Test

The PPI test was conducted with a PPI apparatus (SR-LAB, San DiegoInstruments, San Diego, Calif.) consisting of a startle chamber equippedwith various programming acoustic stimulations. After acclimation, themouse was gently placed in the startle chamber for a 4 min acclimationperiod with a background noise of 65 dB, which continued throughout thewhole PPI test session. One PPI test session consisted of 4 types ofstartle trials, including the trial with the startle pulse (115 dB)alone (PULSEALONE; 115 dB, 20 ms), two trials with the startle pulsepaired with 71 and 77 dB prepulses, respectively (PREPULSE+PULSE; 71dB+115 dB and 77 dB+115 dB), and the trial without stimulus (NOSTIM;background 65 dB only). A test session started and ended, respectively,with four NOSTIM trials and four PULSEALONE trials. In between, each ofthe four types of trials was presented 14 times randomly, that is, 56trials were given in a test session. The intertrial interval was givenrandomly from 5 to 20 s. In the PREPULSE+PULSE trial, a 71 or 77 dBprepulse was given 120 ms before the 115 dBpulse. The magnitude of PPI(PPI %) was determined, after summarizing the startle responses inPULSEALONE and PREPULSE+PULSE trials, according to the equation(PULSEALON−PREPULSE+PULSE)/PULSEALONE×100%. The tested compound orvehicle was given to the animal for 15 min, followed by methamphetamine(2 mg/kg). The PPI test was conducted 10 min after injection ofmethamphetamine. Data were expressed as the mean±S.E.M. Statisticalcomparisons among groups were analyzed by ANOVA with Tukey post hoctest, and differences between groups were analyzed by Student's t-test.Two-way ANOVA with Bonferroni's post hoc test was used to analyzedifferences in the time courses of locomotor activity among groups.Differences were considered significant if P<0.05.

Locomotor Activity

The locomotor activity of the mouse was measured by its interruptions ofinfra-red photobeans in a locomotor cage (42 cm×42 cm×36 cm) in thephotobeam activity system (San Diego Instruments, San Diego, Calif.).After acclimation, the mouse was treated with ci extract or vehicle for15 min, followed by ma (2 mg/kg). Then, the mouse was gently placed inthe locomotor cage and the horizontal and vertical interruptions werecounted every 5 min for 60 min. Total locomotor activity was the sum ofinterruptions within 60 min.

The Rotarod Test

The motor coordinating activity of the mouse was measured by itsperformance on a rotarod in the sdi rotor-rodtm system (San DiegoInstruments, San Diego, Calif.). The mouse was trained four times a dayfor 3 days until it could stay on the rotating drum at a rotating speedof 24 rpm for at least 120 sec. Then, the mouse was subjecting to therotarod test at the rotating speed accelerating gradually from 0 to 30rpm, and the latency to fall from the rotating drum was recorded. Thecut-off time for the latency to fall was 300 sec. Animals were treatedwith the tested drug or vehicle for 15 min before receiving the test.

The Grip Strength Test

Forelimb grip strength was measured by the grip force of forepaws of themouse using the sdi grip strength system (San Diego Instrument, SanDiego, Calif.). The grip strength was recorded by the maximum of threepermissible readings (in grams). The test was conducted every 2 min forthree times, and the averaged grip strength was recorded. Animals weretreated with the tested drug or vehicle for 15 min before the test.

Apomorphine-Induced Stereotypy Behaviors

The mouse was placed into a lidded cylindrical cage (diameter: 12 cm;height: 14 cm) made with 1 cm-separated metal grids for 1 hour'shabituation. Then, the mouse received apomorphine (1 mg/kg, s.c.)Injection. The behaviors of the mouse were videotaped, and thestereotypy behaviors were scored every 5 min for 1 min, starting from 15min before apomorphine injection until 60 min after injection.

The stereotypy behaviors induced by apomorphine were scored by referenceto a previous report (Park et al. 2003) with modifications as thefollowings: 0: normal behaviors (sleeping, normal sniffing); 1:increased activity and sniffing (grooming, rearing, paw licking,jumping); 2: occasional clinging to the side wall of the cage withforepaws; 3: intermittent clinging to the top lid of the cage with allfour paws; 4: uninterrupted climbing with all four paws to the top lid.The highest rating score during the 1 min-recording period was counted.

The Elevated Plus Maze (epm) Test

The epm test was conducted in a plexiglas maze apparatus (San DiegoInstruments, San Diego, Calif.), consisting of a central platform (5×5cm) with four arms (30×5 cm); two open and two closed arms with 25cm-high sidewalls. The maze is elevated 38.5 cm from the room's floor.The mouse was placed at the central platform of the maze facing one ofopen arms and its behaviors in the maze were video-recorded and analyzedevery 5 min-test session by the ethovision video tracking system (noldusinformation technology, wageningen, netherlands). The time spent foreach mouse in the open or closed arm or the central platform as well asthe distances traveled in the open or closed arm were measured. Thedegree of anxiety was accessed by the ratio of the time spent or thedistance traveled in two open arms in a 5 min-test session.

Sedation Assessment

The degree of sedation was measured by the latency for a mouse tocompletely step down from a slightly (3 cm) raised plastic platform(11×8 cm) by reference to a previous report (Horan et al. 1991). Themouse was placed on the platform 15 min after receiving i.p. Injectionof the tested drug. The latency (in sec) for the mouse to step down theplatform with all four paws was recorded. The baseline latency of eachmouse before drug treatment was measured and, if longer than 15 sec, themouse was discarded from the study. A cut-off time of 60 sec was takenas the maximal sedative effect. The percentage of sedation wascalculated by the formula: sedation %=(test latency−baselinelatency)/(60−baseline latency)×100%.

Bilateral Intra-Cerebellar (i.cb.) Microinjection

Mice were anaesthetized with sodium pentobarbital (60 mg/kg i.p.) Andplaced in a stereotaxic frame keeping the bregma-lambda axis horizontal.After shaving the hair and exposing the skull surface, the mouse wasimplanted with two 24-gauge stainless-steel guide cannulas,respectively, directing towards the right and left lateral cerebella(−6.4 mm caudal, ±1.5 mm lateral, −1.0 mm ventral from bregma) accordingto the stereotaxic coordinate of the mouse (Paxions 2001). The cannulaswere fixed on the top of the skull by stainless steel screws and dentalcement. The animal was allowed to fully recover after surgery for atleast one week before the behavioral test was conducted.

On the day for behavioral tests, a 30-gauge injection cannula connectedto a 1 μL hamilton syringe was inserted into the guide cannula for druginjection. The drug solution of 0.5 μL in each side was slowly infusedwith a microinfusion pump (kds311, kd scientific inc.) For 30 s with afurther “hold” time for 60 s. The microinjection site was confirmed bythe positive staining of trypan blue, which was injected through thecannula after behavioral tests. Data from mice with offsite injectionswere discarded.

Methamphetamine and apomorphine were dissolved in normal saline for i.p.And s.c. Injections, respectively. Tested compounds and haloperidol,when given by i.p. Injection, were dissolved in a vehicle containing 20%dmso, 20% cremophor® el (polyoxyethylene castor, sigma-aldrich) and 60%normal saline. Furosemide was dissolved (20 nmole/μL) indimethylsulfoxide for intra-cerebellar microinjection the i.p. or s.c.Injection volume was 10 mL/kg.

The Migraine Model: Capsaicin (i.c.)-Induced Neuronal Activiation In theTCC

Intra-cisteral instillation of capsaicin. Under anesthesia with chloralhydrate (400 and 100 mg/kg, i.p., respectively, for inducing andmaintaining), the rat received a midline skin incision from theoccipital protuberance to the upper cervical area. After catheterizationwith a catheter (PE-10, SIMS Portex Ltd, Hythe, UK) inserted 3 mm deepinto the cisterna magna, the rat was placed in a prone position for 6 h.Then, the capsaicin solution (10 nmol, 100 μL) was instilled through thecatheter into the cisterna magna over 1 min. The rat was then placed ina reverse Trendelenburg position (−30 degrees) for 30 min in order tofacilitate capsaicin distribution within the subarachnoid space,followed by the prone position for another 90 min. Capsaicin (SigmaChemical, St. Louis, MO, USA) was dissolved in the vehicle solutioncontaining 10% ethanol and 10% Tween 80, and sonicated for 5 min, andthen further diluted (1:100) in normal saline as a stock solution storedat 4° C. For the control group, 100 μL of the vehicle was administeredby i.c. instillation. Valproic acid at the dosage of 30 and 100 mg perkg or topiramate at 10, 30, and 100 mg per kg, dissolved in normalsaline, were given at 30 min before caspsaicin administration forpretreatment.

TCC brain sections. Two hours after capsaicin instillation, the rat waseuthanized by an overdose of chloral hydrate (1.6 g/kg, i.p.) and thenperfused via the ascending aorta with 150 mL 0.5% sodium nitrate,followed by a 500 mL fixative containing paraformaldehyde (4%) in 0.1 Mphosphate buffer (PB, pH 7.4). The brainstem with attached cervical cordwas dissected, stored overnight in the perfusion fixative, and thendehydrated with 20% and 30% sucrose, in series. Brainstem and uppercervical spinal cord sections (50 μm) were serially sectioned using acryostat (LEICA CM3050S, Nussloch, Germany) from 1 mm rostral to theobex to the C6 level of the spinal cord.

Immunohistochemistry of c-Fos protein in TCC sections. Free-floatingimmunohistochemistry of c-Fos protein was conducted using theavidin-biotin method. Briefly, TCC sections were incubated in 1% NaBH4in 0.1 M PB for 20 min, rinsed thrice with 0.1 M PB for 10 min, andtreated with 0.5% hydrogen peroxide in 0.1 M PB for 30 min. The sectionswere then washed thrice with 0.1 M PB for 10 min, followed by 1 hourincubation with 0.1 M PB containing 5% goat serum and 0.2% triton X-100.The sections were incubated with an anti-c-Fos rabbit polyclonalantibody (Calbiochem, San Diego, Calif., USA.) in 1:7000 dilution with0.1 M PB containing 5% goat serum and 0.2% triton X-100 at 4° C. for 48h. After a 10-min rinse with 0.1 M PB thrice, the sections wereincubated with biotinylated anti-rabbit IgG (Vector Labs, Burlingame,Calif., USA) in 1:200 dilution with 0.1M PB containing 5% goat serum and0.2% triton X-100 for 2 h at room temperature. The sections were thenwashed thrice with 0.1 M PB for 10 min and incubated with horseradishperoxidase avidin D (Vector Labs, Burlingame, Calif., USA) in 1:500dilution with 0.1M PB containing 5% goat serum and 0.2% triton X-100 for1 h in the dark at room temperature. Immunoreactions were visualizedusing the DAB Reagent kit (KPL, Gaithersburg, Md., USA).

Measurement of the total number of C-fos-ir neurons in the TCC. C-Fos-irneurons, i.e., neurons with stained nuclei, were counted under amicroscope (Olympus BX51, Essex, UK) by an observer blinded to thetreatment group. Data were confirmed in randomly selected sections by asecond investigator who was also blinded to the treatment group. Thetotal number of c-Fos-ir TCC neurons were estimated based on theformular derived in our previous study (Fan et al., 2012):16(N1+N2)/2+53(N2+N3)/2, where N1, N2, and N3 were the c-Fos-ir neuronalnumbers measured at 0.6, −1.2, and −9 mm from the obex, respectively.

Example 3 Effects of α6-Selective Pyrazoloquinolinones in an AnimalModel Mimicking Neuropsychiatric Disorders with Sensorimotor GatingDeficit

We first examined the effects of two pyrazoloquinolinone compounds onthe impairment of PPI induced by METH, an animal model mimickingneuropsychiatric disorders with sensorimotor gating deficit. Thecompounds were compound 6(7-methoxy-2-(4-methoxyphenyl)-2Hpyrazolo[4,3-c]quinolin-3(5H)-one) thatselectively modulates α6-containing receptors, and compound 11(8-chloro-2-(4-methoxyphenyl)-2Hpyrazolo[4,3-c]quinolin-3(5H)-one) thatalso modulates α6-containing receptors to an even higher extent but inaddition strongly modulates other GABA_(A) receptor subtypes. We foundthat both compounds 6 and 11 when given by i.p. injection at 10 mg/kgsignificantly rescued METM-impaired PPI (FIG. 4). Furthermore, theinhibition of this beneficial effect by furosemide provides strongevidence for the involvement of α6-containing receptors, becausefurosemide is an α6-preferring GABA_(A) receptor negative modulator(FIG. 5).

Example 4 Compounds with Improved Metabolic Properties Based on“Compound 6”

Metabolic pathways that may disrupt the activity of compounds with arylmethoxy groups were investigated. It was found that deuterated analogsgreatly enhanced the half-life of compounds in rat and human livermicrosomes. Compound 6 was found to have a half-life of 175 minuteswhile the deuterated analog RV-I-29 has a half-life of >3000 minutes.This finding is crucial as drugs with a high rate of metabolism likelyhave a significantly shorter window of activity in vivo (FIG. 21).

Deuterated analogs of compound 6 (including 1-29 and DK-56-1) weretested in the same PPI model as above and found to have similar activityto the non-deuterated analog (FIG. 6), however duration of drug was nottested in these experiments. Experiments to test duration of the drug invivo are currently underway.

Example 5 Additional α6 Selective Pyrazoloquinolinones

FIG. 4 shows that LAU 463 and LAU 159 displayed a comparable rescueeffect as Compound 6 in METH-impaired PPI.

Example 6 Search for Water Soluble α-6 Bz/GABA-A Receptor SelectiveLigands

Compound 5 and compound 6 were identified as selective α6β3γ2 receptors.Many different analogues of compound 6 were synthesized and screened toincrease the water solubility of the compounds to facilitate theirapplication in vivo.

Compounds were pharmacologically tested for selective activity forα6-subunit-containing receptors.

Several of the compounds exhibited increased water solubility and wereinvestigated for their receptor subtype-selectivity. The ligandsscreened were sufficiently potent for testing their effectiveness invarious animal models of CNS disorders. More potent and solublecandidates that are α6β3γ2 GABA-A receptor selective will be developed.These compounds may be used to develop new therapies for various CNSdiseases.

Example 7 Solubility of Compounds

The solubility of the compounds was examined by sonicating 2 mg of eachrespective compound in 2 mL of DI water at 60° C. for 2 hours. Solutionswere cooled to room temperature and then filtered using a syringefilter. The resultant solutions were injected into a mass spectrometerusing a SIM method, calibrated to the respective retention times andmass units of each compound. Calibration curves using standard solutions(5 data points) of each compound dissolved in methanol were used tocalculate the unknown aqueous concentrations of each compound. Thesolubilities of the compounds are shown in TABLE 1.

TABLE 1 Solubility of compounds. OCD3 Compounds

DK-I-58-1 LAU 463 MW: 373.22 Solubility: 9.2 ng/μL

DK-I-59-1 LAU 159 MW: 328.77

DK-I-56-1 Comp 6 “Mono D” MW: 324.35 Solubility: 11.4 ng/μL

DK-I-60-3 Comp 6 “Di D” MW: 327.34 Solubility: 16.0 ng/μL

DK-I-86-1 MW: 325.32 Solubility: 211 ng/μL

Example 8 Electrophysiological Experiments with Xenopus Oocytes

Mature female Xenopus laevis (Nasco, Fort Atkinson, Wis., USA) wereanaesthetized in a bath of ice-cold 0.17% Tricain (Ethyl-m-aminobenzoat,Sigma-Aldrich, St. Louis, Mo., USA) before decapitation and transfer ofthe frog's ovary to ND96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mMHEPES; pH 7.5). Following incubation in 1 mg/mL collagenase(Sigma-Aldrich, St. Louis, Mo., USA) for 30 min, stage 5 to 6 oocyteswere singled out of the ovary and defolliculated using a platinum wireloop. Oocytes were stored and incubated at 18° C. in NDE medium (96 mMNaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, 1.8 mM CaCl2; pH 7.5) that wassupplemented with 100 U per mL-1 penicillin, 100 μg per mL-1streptomycin and 2.5 mM pyruvate. Oocytes were injected with an aqueoussolution of mRNA. A total of 2.5 ng of mRNA per oocyte was injected.Subunit ratio was 1:1:5 for αxβγ2 (x=1,2,3,5) and 3:1:5 for α4/6β3γ2 andαβδ receptors. Injected oocytes were incubated for at least 36 h beforeelectrophysiological recordings. Oocytes were placed on a nylon-grid ina bath of NDE medium. For current measurements, the oocytes were impaledwith two microelectrodes (2-3MΩ), which were filled with 2 M KCl. Theoocytes were constantly washed by a flow of 6 mL per min-1 NDE thatcould be switched to NDE containing GABA and/or drugs. Drugs werediluted into NDE from DMSO solutions resulting in a final concentrationof 0.1% DMSO. Maximum currents measured in mRNA injected oocytes were inthe microampere range for all receptor subtypes. To test for modulationof GABA induced currents by compounds, a GABA concentration that wastitrated to trigger 3-5% of the respective maximum GABA-elicited currentof the individual oocyte (EC3-5) was applied to the cell together withvarious concentrations of tested compounds. All recordings wereperformed at room temperature at a holding potential of −60 mV using aWarner OC-725C TEV (Warner Instrument, Hamden, Conn., USA) or a DaganCA-1B Oocyte Clamp or a Dagan TEV-200A TEV (Dagan Corporation,Mineapolis, Minn., USA). Data were digitized using a Digidata 1322A or1550 data acquisition system (Axon Instruments, Union City, Calif.,USA), recorded using Clampex 10.5 software (Molecular Devices,Sunnyvale, Calif., USA), and analyzed using Clampfit 10.5 and GraphPadPrism 6.0 (La Jolla, Calif., USA) software. Concentration-response datawere fitted using the Hill equation. Data are given as mean±SEM from atleast three oocytes of two batches.

For selected test compounds full dose response curves were recorded inα1-6β3γ2 receptors. For selected compounds, dose response curves inadditional receptor subtypes (α1, 4, 6β3δ, α6β1, 2γ2, α1-6β3) were alsoobtained. For a wider panel of compounds, a two point screeningpharmacology was obtained at 1 and 10 μM compound concentrations.

Compounds were grouped into “parent compounds” (the pyrazoloquinolinones“compound 6”, LAU 463, LAU 159, and LAU 165); their deuteratedderivatives; and compounds with novel chemotypes. Compound 6, LAU 463,and LAU 159 all displayed among αβγ2 receptors a pronounced selectivityfor the α6β3γ2 receptor, and modulated EC3-6 GABA currents strongly withmaximum efficacies ranging from ˜400% (LAU 159) to ˜1000% enhancement ofthe reference current (FIG. 2 and FIG. 3). In α1,4,6β3δ receptors, theyalso display α6 preference, but displayed lower efficacy (below 400% atour experimental conditions) (FIG. 2 and FIG. 3).

The primary objectives of the large functional screening shown in FIG.24 were the following:

(1) Test whether deuterated analogues are as active as the parentcompounds in the receptor of interest, the α6β3γ2 receptor. This was thecase (within the low precision of the method) for all deuteratedcompounds.

(2) Verify that the deuterated analogues were as selective as the parentcompounds. For all deuterated analogues of these α6 preferring positivemodulators we investigated effects at 1 and 10 μM in a series ofscreening experiments to determine if their efficacy and selectivity iscomparable to the parent compounds (FIG. 24). Overall, we obtainedsatisfactory results for most deuterated analogues, i.e., they displayactivities very similar to their structural parents.

(3) Test new compounds and select them for further analysis if activityin the α6β3γ2 receptor was observed. Bioisosteric compounds withheteroatoms in the core scaffold and with analogous substituents ascompound 6 (CW-03-030a and DK-I-86-1) were thus investigated. The reasonfor this is the poor solubility of pyrazoloquinolinones, which can beimproved by introducing heteroatoms into the backbone of theheterocyclic core. Preliminary screening of CW-03-030a and DK-I-86-1(FIG. 24) led to a follow up on the more promising DK-I-86-1. Whilemaximum efficacy was lower than for the lead compounds, the selectivitywas very good and if bioavailability is superior, the compound may be asgood as or better than compound 6 for in vivo studies.

(4) Characterize candidate negative control compounds, which should beinactive in all receptors. Here we identified a putative negativecontrol compound. LAU 165 and its deuterated analogue (DK-I-87-1) wereinactive in all receptors that were tested so far (FIG. 24). Thus, thesecompounds can potentially be used in further in vivo studies as controlcompounds to demonstrate that inactive compounds of the same chemotypeexist, and thus a specific substitution pattern is necessary for both invitro and in vivo specific activity.

As shown in FIG. 24, in αxβ3γ2 receptors, the parent compounds compound6, LAU 463 and LAU 159 and their deuterated analogues are (efficacy-)selective for α6β3γ2 receptors over the other αxβ3γ2 receptors. Inbinary αxβ3(x=1,2,3,5) receptors, they display low efficacy as well. Inβ1-containing receptors, data suggests loss of α6-preferences, andincreased activity in α1β1-containing receptors compared toα6β1-containing receptors. This observation needs follow up studies, seebelow.

LAU 165 and the deuterated analogue DK-I-87-1 were shown to beessentially inactive in all tested receptors.

Values at 100 nM and 1 μM reflect modulations that can be expected tooccur in vivo, based on the brain free fraction concentrations that weredetermined (see FIGS. 17 and 18).

Additional pharmacological experiments. Selected compounds of thoselisted in FIG. 24, as well as additional compounds will be tested alsoin receptors subtypes containing the β1 and β2 subunits to investigatehow alpha selectivity and beta selectivity combine together into asubtype specific response covering all three beta isoforms. Furthermore,pharmacological testing in delta-containing receptors will also beconducted in more depth. The inactive compound DK-I-87-1 will be testedin co-application experiments with active compound DK-I-56-1 to clarifyif it is a non-binder or a silent binder (null modulator) of the bindingsite used by DK-I-56-1. We keep the option to test selected compoundsalso in other cells, such as mammalian cells, expressing recombinantreceptors if needed to substantiate any claims on efficacy orselectivity. We also optionally test in human recombinant receptorsrather than the so far used rat receptors. Furthermore, selectedcompounds (preferentially these that are active in disease modelexperiments) can also conceivably be tested electrophysiologically incultured neurons, or in slices of α6-expressing tissues such ascerebellum, cochlear nucleus, trigeminal ganglion, to study the effectsof compounds on native receptors.

Additional experiments to evaluate effect of compounds at GABA_(A)receptors in dorsal root ganglia: Dorsal root ganglia and trigeminalganglia from naive and neuropathic pain model rats, where bothtrigeminal nerve lesions and ischiatic nerve lesions can be employed,will be prepared and worked up immunohistochemically and biochemicallyusing antibodies directed against α6-subunits to explore the expressionlevel of this subunit under normal and pathological conditions. In casethat α6 subunits can be detected in dorsal root ganglia as well, followup studies will be initiated to test effectiveness of the compounds inrodent models of neuropathic pain of spinal nerves.

Example 9 Effects of Subchronic Treatment with DK-I-56-1 in IoN-CCl Rats

Chronic constriction injury (CCl) is a common neuropathic pain model.The unilateral ligature of the infraorbital nerve (IoN) in rats, as ananimal model of peripheral neuropathic pain, and more specifically,trigeminal neuralgia and trigeminal neuropathy, was performed asdescribed by Desuere and Hans (Deseure et al. J. Vis. Exp. 2015, 103,e53167). Wistar rats (46 in total) were randomly assigned to one of fourexperimental groups and then were behaviorally tested in a blindedmanner. For studying the effects of subchronic treatment with thenanoemulsion formulation of DK-I-56-1 in IoN-CCl rats, DK-I-56-1 (10mg/kg i.p.) or its vehicle (placebo nanoemulsion i.p.) were injecteddaily for 14 consecutive days after surgery. Sham-operated rats weretreated in parallel. By a procedure slightly modified from Djordjevic etal. (Int. J. Pharm. 2015, 493, 40-54), biocompatible nanoemulsions wereprepared by high pressure homogenization at 50° C. They were composed ofmedium chain triglycerides, castor oil, soybean lecithin, sodium oleate,polyoxyethylensorbitan monooleate, butylhydroxytoluene, DMSO andultra-pure water. Treatment was administered at 3:00 PM, after finishingthe behavioral testing or handling/habituation for the given day. Twotypes of behavioral tests in single observation cages were performedrepeatedly, on separate days. First, the response to the application ofa graded series of three von Frey filaments onto ipsilateral vibrissalpad was measured on pre-operative days −3 and −1 and post-operative days7, 14, 21, and 28. The response was categorized as score 0 (a completelack of response), 1 (a stimulus detection), 2 (a withdrawal reaction),3 (an escape/attack response), or 4 (asymmetric face grooming). Theother parameter, face grooming during body grooming, was manuallyassessed during 10 min of recording on pre-operative day −2 andpost-operative days 2, 4, 8, and 15 (i.e. after 1, 3, 7, and 14applications of treatment). This parameter represents a reliableethological measure to control for possible confounding by nonspecifictreatment effects, such as sedation or motor impairment.

The results of the assessment of central neuropathic pain are summarizedin FIG. 14A (response score to von Frey filament stimulation for allfour groups) and FIG. 14B (only for clarity, response score for twogroups of IoN-CCl rats). The criterion for exclusion of animals fromstatistical analyses was predefined for Ion-CCl-Placebo group(consistently decreased reactivity in post-surgery period when comparedto the pre-surgery values; 1 animal excluded), and for Sham-Placebogroup (consistently increased reactivity in post-surgery period whencompared to the pre-surgery values, the mean difference being more thanor equal 1.25 score points; 2 animals excluded); the animals treatedwith DK-I-56-1 could not have been excluded.

As shown in FIG. 14A, basal response scores (pre-operative day −1) weresimilar among four groups, while the scores of two IoN-CCl groups werevirtually the same. Seven days after surgery and afterwards, theresponse score in the IoN-CCl-Placebo group was consistently increased,at the level of 2.4 score points and above, demonstrating a nociceptiveresponse to the application of stimuli. Repeated treatment withDK-I-56-1 provided a preventive effect on the development of neuropathicpain, and this anti-allodynic effect was statistically significant onpost-operative days 14 and 21. On the post-operative day 28, i.e. 14days after the last dose of DK-I-56-1, the significance of the effect ofDK-I-56-1 disappeared (FIG. 14B). Thus, DK-I-56-1 demonstrated acapability to be applied as a prophylactic measure for the management ofneuropathic pain related to the impairment of branches of the trigeminalnerve.

The results of the assessment of face grooming during body grooming, asa physiological manifestation of the rat's behavior on which IoN-CCl haslittle or no effect, are summarized in FIG. 15. The level of activitywas distinct on all of the five days measured, and no significantdifferences among groups were detected. This parameter, characterized bya substantial variability, demonstrated the lack of sedation-like ormotor-impairing influences of DK-I-56-1, at least at the time ofbehavioral testing. Moreover, in a separate pilot experiment, DK-I-56-1acutely administered in the dose range 10-55 mg/kg did not impairrotarod performance of male Wistar rats when tested 20 min aftertreatment administration.

As trigeminal neuralgia and trigeminal neuropathy are chronic types ofpain which usually require long-term pharmacotherapy, it is planned toperform an additional experiment, in male Wistar rats subjectedexclusively to IoN-CCl. After assessment of the neuropathy (mechanicalallodynia to von Frey filaments) on Day 21, the rats would be randomlyassigned to three groups: placebo, DK-I-56-1, or DK-I-87-1, the compoundinactive at α6-containing GABA_(A) receptors. The respective treatmentwould be applied i.p. once daily during 14 days, starting on Day 21.Possible acute effects of the treatment would be estimated on Day 22,while the potential of eliciting chronic analgesic effects would beassessed on post-surgery days 28 and 35. Statistical analysis for eachof measurement times would be performed by one-way ANOVA followed byStudent Newman Keuls test.

Example 10 Pharmacokinetic Behavior of Compound 6, DK-I-56-1, andDK-I-86-1, in Rats and of DK-I-56-1 in Mice After an Acute Treatmentwith the Nanoemulsion and/or Suspension Formulation

Five pharmacokinetic studies have been performed; four in male Wistarrats and one in male Swiss mice. In experiments in rats, single 10 mg/kgdoses of compound 6, DK-I-56-1 or DK-I-86-1 were administered i.p. tothree rats per each of five time points. At predetermined timeintervals, i.e., 5, 20, 60, 180, and 720 min after dosing, the blood andbrain samples of the rat were collected. In the experiment in mice,single 10 mg/kg or 30 mg/kg doses of DK-I-56-1 were administered i.p. tothree mice per each of 5 time points. At predetermined time intervals,i.e., 5, 20, 40, 120, and 360 min, the blood and brain samples of themouse were collected. In rats, two experiments, with compound 6 andDK-I-56-1, were carried out with the 2 mg/mL formulation of ananoemulsion (as described in Example 10), given at an injection volumeof 5 mL/kg. The other two experiments in rats, with DK-I-56-1 andDK-I-86-1, were performed with the suspension formulation, given at aninjection volume of 2 mL/kg (the ligands were suspended with the aid ofsonication in solvent containing 85% distilled water, 14%propyleneglycol, and 1% Tween 80). The experiment in mice was performedwith the 2 mg/mL nanoemulsion formulation of DK-I-56-1. Concentration ofcompound 6, DK-I-56-1 or DK-I-86-1 extracted from the respective samplesby solid phase extraction was determined by ultra-high performanceliquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). In aseparate in vitro experiment, the rapid equilibrium dialysis assay wasused to determine free fraction of DK-I-56-1 in rat plasma and braintissue.

The results of the assessment of pharmacokinetic behavior of compound 6,DK-I-56-1 and DK-I-86-1 are summarized in FIG. 16-20 , which representconcentration-time curves, measured as total concentration in plasma(ng/mL) and brain (ng/g), together with the calculated pharmacokineticparameters in plasma and brain. In the case of DK-I-56-1 in rats (FIGS.17 and 18), the estimated free concentrations in brain tissue were alsopresented.

To ease comparison between the concentrations obtained in vivo and thoseused in vitro, the description of the kinetic results refers to molarrather than weight concentrations. In rats, compound 6 (FIG. 16) andDK-I-86-1 (FIG. 19) reached high submicromolar concentrations in braintissue. Such relatively low total brain concentrations attained afteradministration of the 10 mg/kg dose of compound 6 and DK-I-86-1 in asuspension cannot be solely ascribed to formulation issues, given thatDK-I-56-1 reached micromolar concentrations in rat brain tissue afteradministration of the same dose of both, the nanoemulsion formulation(FIG. 17) and the suspension formulation (FIG. 18). While free fractionof DK-I-56-1 in rat blood is exceptionally low (below 0.5%), its freefraction in rat brain tissue equals 19.45%, which enables reachingpharmacologically relevant free concentrations in the range of 100 nMand above. With submicromolar total concentrations in mouse brain,kinetic behavior of DK-I-56-1 dosed at 10 mg/kg was less favorable foreliciting central nervous system effects in mice than in rats.Nonetheless, the maximum concentration of DK-I-56-1 attained in mousebrain after the dose of 30 mg/kg was as high as 7.5 μmol/L (FIG. 20);this value demonstrates the dose-dependency of brain tissue kinetics ofthis ligand. Moreover, brain tissue kinetics of DK-I-56-1 is relativelyslow, especially in rats, with elimination half-life being approximately4 h, and time of maximum concentration 3 h. It is worthy of noting thatDK-I-86-1 had the longest half-life in rat brain tissue (approximately 9h). It can be concluded that all four ligands reached substantialmicromolar maximum concentrations in plasma of both, the rats and themice, while their penetration into the central nervous system wasslightly restricted, given the consistently moderately lowerconcentrations in brain when compared to those attained in plasma.Nevertheless, the kinetic studies in rodents have shown that theseligands, and especially DK-I-56-1, possess pharmacokinetic propertiesbeneficial for the proposed neuropsychopharmacological application.

Example 11 Examination of α6-GABA-A Receptor as Target forNeuropsychiatric Syndrome

We found the intractable motor tics in a pediatric patient were subsidedwithin 1 hour after taking the leaf extract of a local herb,Clerodendron inerme Gaertn (Cl) (Fan et al., 2009). We then identifiedan active constituent, hispidulin, from the ethanol extract of Clleaves, which is a positive allosteric modulator (PAM) of GABA-Areceptors, including the α6 subunit-containing GABA-ARs (α6 GABA-ARs).Since hispidulin is not selective for α6 GABA-AR, we obtained the firstselective α6 GABA-AR PAM, compound 6. Compound 6, like KLP-1,effectively rescued the impairment of prepulse inhibition of the startleresponse (PPI) induced by methamphetamine, a measurement of thesensorimotor gating function that is deficit in several neuropsychiatricdisorders, including tic disorders and schizophrenia.

We further conducted a comprehensive study examining the effects ofcompound 6 in various behavioral models mimicking tic disorders,including the hyperlocomotion induced by methamphetamine (a dopaminereleaser), stereotypy climbing behaviors induced by apomorphine (adopamine receptor agonist). Using anatomical and pharmacologicalapproaches by intracerebellar microinjection and pharmacologicalapproaches using furosemide as a non-competitive antagonist anddiazepam, that does not act at α63 g2 GABA-ARs, we also confirmed thatα6 GABA-AR in the cerebellum is the action target of Compound 6 (FIG.5).

Intraperitoneal (i.p.) injection of Compound 6 significantly rescuedmethamphetamine-induced hyperlocomotion. This effect was significantlyantagonized by intra-cerebellum microinjection of furosemide, aselective α6GABA_(A)R antagonist (FIG. 7). On the other hand, Compound 6(i.p.) did not affect stereotypy climbing behaviors induced byapomorphine, a dopamine receptor agonist. (FIG. 8). However, Compound 6did not affect the spontaneous motor activity (FIG. 7, the left pairedbars), motor coordination in the rotarod test (FIG. 10), or the gripstrength (FIG. 9), neither displayed any significant sedation (FIG. 11)or anxiolytic activity (FIG. 12) in the sedation assessment and elevatedplus maze test, respectively. These results suggest that compound 6 cansuccessfully pass through the blood brain barrier to rescuemethamphetamine-induced hyperlocomotion possibly via enhancingcerebellar inhibitory control on the striatel dopaminergic activitythrough positively modulating α 6GABA_(A)Rs in cerebellar granule cells,but not affect dopamine receptor response directly. It is also suggestedthat the α6GABA_(A)R in the cerebellum is a new target for the treatmentof TS or tic disorders. The PAM selective to α6GABA_(A)Rs may be a novelclass of anti-tic therapy.

Example 12 The Effect of Compound 6 on i.c. Capsaicin-Induced TNCNeuronal Activation, an Animal Model Mimicking Migraine

FIG. 13 shows that Compound 6 when given by i.p. injection at 10 and 30mg/kg significantly decreased the number of c-Fos-ir neurons in the TNC,a measurement of the number of activated TNC neurons, induced by i.c.capsaicin. This suggests that systemic administration of Compound 6reduces TNC neuronal activation and has the potential for migrainetreatment.

Example 13 Microsomal Stability Assay

4 μL of 1 mM test compound at a final concentration of 10 μM dissolvedin DMSO/ACN/Methanol/Ethanol was preincubated at 37° C. for 5 minutes ona digital heating shaking dry bath (Fischer scientific, Pittsburgh, Pa.)in a mixture containing 282 μL of water, 80 μL of phosphate buffer (0.5M, pH 7.4), 20 μL of NADPH Regenerating System Solution A (BDBioscience, San Jose, Calif.) and 4 μL of NADPH Regenerating SystemSolution B (BD Bioscience, San Jose, Calif.) in a total volume of 391.2μL. Following preincubation, the reaction was initiated by addition of8.8 μL of either human liver microsomes (BD Gentest, San Jose, Calif.),mouse liver microsomes (Life technologies, Rockford, Ill.), at a proteinconcentration of 0.5 mg/mL. Aliquots of 50 μL were taken at timeintervals of 0 (without microsomes), 10, 20, 30, 40, 50 and 60 minutes.Each aliquot was added to 100 μL of cold acetonitrile solutioncontaining 1 μM/2 μM internal standard. This was followed by sonicationfor 10 seconds and centrifugation at 10,000 rpm for 5 minutes. 100 μL ofthe supernatant was transferred into Spin-X HPLC filter tubes (CorningIncorporated, NY) and centrifuged at 13,000 rpm for 5 minutes. Thefiltrate was diluted 100 fold and subsequently analyzed by LC-MS/MS withShimadzu LCMS 8040, (Shimadzu Scientific Instruments, Columbia, Md.).The ratio of the peak areas of the internal standard and test compoundwas calculated for every time point and the natural log of the ratiowere plotted against time to determine the linear slope (k). Themetabolic rate (k*C₀/C), half-life (0.693/k), and internal clearance(V*k) were calculated, where k is the slope, C₀ is the initialconcentration of test compound, C is the concentration of microsomes,and V is the volume of incubation in μL per microsomal protein in mg.All experiments were repeated three times in duplicates. Results areshown in TABLE 2.

TABLE 2 Half-life of compounds. Half-life (min) % left after 1 Half-life(min) % left after 1 Compound (HLM) hr. (HLM) (MLM) hr. (MLM) DK-I-58-1 204.6 ± 10  81.5 ± 0.11   140 ± 7.18  73.8 ± 0.15 LAU 463   104 ± 466.86 ± 0.13 113.17 ± 5.15   69 ± 0.16 DK-I-59-1   649 ± 81   93 ± 0.09165.74 ± 7.5  76.6 ± 0.11 LAU 159 206.77 ± 15 81.44 ± 0.16  89.07 ± 5.2259.83 ± 0.23 DK-I-56-1   520 ± 51 91.69 ± 0.09  628.8 ± 73  92.8 ± 0.09Comp 6  212.5 ± 25  86.1 ± 1.0 194.26 ± 11.41 80.35 ± 0.14 DK-I-60-3780.73 ± 238 91.51 ± 0.18 846.85 ± 276 93.08 ± 0.19 RV-I-029   2972 ±742 95.35 ± 1  857.8 ± 235 93.17 ± 016 DK-I-93-1   640 ± 126  92.2 ±0.15   263 ± 5.96 85.53 ± 0.07 Comp 11  135.4 ± 5  72.5 ± 0.11  115.6 ±5.2  69.2 ± 0.15 DK-I-86-1 752.30 ± 261  92.3 ± 0.13  550.6 ± 78  91.3 ±0.12 DK-II-13-1   794 ± 182   93 ± 0.14  139.2 ± 5.8 74.77 ± 0.12DK-II-58-1   2341 ± 0.001  97.8 ± 0.06   4250 ± 0.001 98.39 ± 0.06DK-II-48-1  727.7 ± 115  93.4 ± 0.11  680.6 ± 114 93.12 ± 0.22DK-II-59-1  725.8 ± 146   93 ± 0.12 250.62 ± 13  85.1 ± 0.10 DK-II-18-1  511 ± 44  91.6 ± 0.10   265 ± 17  85.6 ± 0.12 DK-II-60-1  592.5 ± 96 92.5 ± 0.12   640 ± 102  92.4 ± 0.12 DK-I-87-1   990 ± 159  94.7 ± 0.07  993 ± 189 94.91 ± 0.10

DK-I-58-1 and LAU 463 are bromine containing α6 analogs. The deuteratedanalog is more stable compared to methoxy compound which is nearly 15-20less stable. The chlorine containing non-deuterated analog (LAU 159) isshown to be less stable indicated nearly 80% stability in Human and evenlesser stability (˜60%) in mouse microsomes compared to other compoundsthat have (—OCH₃) in the adjacent position without the chlorine anddeuterated compound with the deuterium 15-20% more stable in bothspecies and similar stability was observed with the DK-I-93-1 and Comp11 analogs with the Deuterium containing analog having the higherstability.

The mono (RV-I-029 and DK-I-56-1) and di deuterated (DK-I-60-3) analogsof Comp 6 have shown a higher stability that is more than 90% comparedto Comp 6 which is 86% stable in Human and 80% stable in Mouse livermicrosomes respectively with significant variation in the mouse species.

DK-I-86-1 and DK-II-13-1 are the pyridine containing compounds. Incomparison, the Deuterated (—OCD₃ containing DK-I-86-1) has significanthigher in the stability (nearly 20%) compared to non-Deuterated(DK-II-13-1) in mouse liver microsomes, however they behave same inHuman liver microsomes. The other pyridine containing analogs with theBromine (DK-II-58-1 and DK-II-48-1) and Chlorine (DK-II-59-1 andDK-II-18-1) behave similarly in the both the species but they have shownsignificant variation from the corresponding compounds that do notcontain the pyridine ring.

The DK-II-60-1 analog with the methoxy substitution in place of brominein DK-II-58-1 behave similar to it and also DK-I-87-1 with the —OCD₃ atthe ortho position compared to the DK-I-59-1 analog has significantvariation in the stability in the mouse liver microsomes but not in thehuman species.

In conclusion, the deuterium containing analogs of Alpha 6 compoundshave a significant variation and have shown higher stability compared tonon-deuterated analogs.

Example 14 Alpha 6 Cytotoxicity Study

Reagents and Instrumentation: Human liver hepatocellular carcinoma(HEPG2) cell line was purchased (ATCC) and cultured in 75 cm2 flasks(CellStar). Cells were grown in DMEM/High Glucose (Hyclone, #SH3024301)media to which non-essential amino acids (Hyclone, #SH30238.01), 10 mMHEPES (Hyclone, #SH302237.01), 5×106 units of penicillin andstreptomycin (Hyclone, #SV30010), and 10% of heat inactivated fetalbovine serum (Gibco, #10082147) were added. Cells were harvested using0.05% Trypsin (Hyclone, #SH3023601), which disrupts the cell monolayerand proteolytically cleaves the bonds between the cells and flask. Thecell viability assay was evaluated using CellTiter-Glo™ Luminescent CellViability Assay Kit (Promega, Madison, Wis.) which contains luciferaseand all its substrate except ATP. The controls for the cytotoxicityassay used were (E)-10-(bromotriphenylphosphoranyl)decyl4-(4-(tert-butyl)phenyl)-4-oxobut-2-enoate (400 μM in DMSO, positivecontrol) and DMSO (negative control). Cell culture was performed in aBaker Company Class II Biological Safety Cabinet. All luminescencereadings were performed on a Tecan Infinite M1000 plate reader. Smallvolume transfers were performed on the Tecan Freedom EVO liquid handlingsystem with a 100 nL pin tool transfer (V&P Scientific). Serialdilutions were done in 96-well polypropylene plates (Corning, #3365) andassays were conducted in 384-well white optical bottom plates.

Luminescence-Based VDR-Mediated Transcription Assay Protocol: After 48hours of incubation at 37 oC with 5% CO2, the cells were harvested with4 mL of 0.05% Trypsin, added to 10 mL of the assay buffer, DMEM/HighModified buffer without phenol red, and spun down for 3 minutes at 1000rpm. The media was removed and cells were resuspended in the DMEM assaymedia. To each well, 40 μL of cells were added to yield a finalconcentration of 15,000 cells per well. The plates were then spun downfor 2 minutes at 1000 rpm. After 2 hours, plated cells were treated with100 nL×4 of small molecules and controls which were added using the pintool. After 48 hours of incubation at 37 oC with 5% CO2, 20 μL of CellTiter-Glo™ Luminescence Assay Kit (cytotoxicity assay) were added andluminescence was read. Controls were measured within each plate todetermine the z′ factor (Equation 1) and to enable data normalization.Three independent experiments were performed in quadruplicate and datawas analyzed using nonlinear regression with variable slope (GraphPrism,Equation 2). The luminescence intensity of control (untreated) cells wastaken as 100% viability, and the relative cell viability compared tocontrol was calculated. Data are presented as the means±SEM of a seriesof three experiments.

$\begin{matrix}{Z^{\prime} = {1 - \left( \frac{\begin{matrix}{3 \times \left( {{{Standard}\mspace{14mu} {Deviation}\mspace{14mu} {Positive}} +} \right.} \\\left. {{Standard}{\mspace{11mu} \;}{Deviation}{\mspace{11mu} \;}{Negative}} \right)\end{matrix}}{{{{Average}\mspace{14mu} {Positive}} - {{Average}\mspace{14mu} {Negative}}}} \right)}} & {{Equation}\mspace{14mu} 1} \\\frac{{Bottom} + \left( {{top} - {bottom}} \right)}{\left( {1 + 10^{{{{{logIC}\; 50} - X})}{({HillSlope})}}} \right)} & {{Equation}\mspace{14mu} 2}\end{matrix}$

Results are shown in TABLE 3, FIG. 17, FIG. 18, and FIG. 19. Thecompounds such as DK-I-58-1, DK-I-59-1, DK-I-56-1, DK-I-60-3, DK-I-86-1,and DK-II-48-1 are non-toxic even at 400 μM. The compounds RV-I-029,DK-I-93-1, DK-I-87-1, DK-I-94-1, DK-II-18-1, and DK-I-90-1 aremoderately toxic and their LD50 values are more than 200 μM.

TABLE 3 Cytotoxicity study results for HEPG2 cell lines. S. No.Non-toxic at 400 μM LD₅₀ values higher than 200 μM 1 DK-I-58-1 RV-I-0292 DK-I-59-1 DK-I-93-1 3 DK-I-56-1 DK-I-87-1 4 DK-I-60-3 DK-I-94-1 5DK-I-86-1 DK-II-18-1 6 DK-II-48-1 DK-I-90-1

Example 15 Rotorod Assay

30 White female Swiss Webster mice were trained to maintain balance at aconstant speed of 15 rpm on the rotarod apparatus (Omnitech ElectronicsInc., Nova Scotia, Canada) until mice could perform for three minutes atthree consecutive time points. Separate groups of mice receivedintraperitoneal (i.p.) injections of vehicle (40% soybean oil, 10%water, 15% kolliphor, and 35% glycerol), α6 test compounds (1, 5, 20, or40 mg/kg), and diazepam as a positive control compound (1 mg/kg or 5mg/kg) in an approximate volume of 100 μL. Ten minutes after eachinjection, mice were placed on the rotarod for three minutes. A fail wasassigned for each mouse that fell from the rotarod prior to 3 minutes.Mice were rested two to three days before administration of another doseor a different compound. The protocol was changed to a 200 μL i.p.injection for vehicle and compounds due to solubility. Results are shownin FIG. 20.

Example 16 Tinnitus Experiment

About 96 rats (Sprague Dawley, 8 weeks old, b.w. 250 to 300 gm) will beused. Animals are randomly divided into 2 groups: (a) group A, loudsound-treated and (b) group B, control. Each group will be housed in alaboratory room. Two groups will be further divided into 3 subgroups toeach receive (a) sound treated and additional injection of vehicle (20%DMSO+20% Cremophor® EL+60% saline, Sigma) (b) an additional injection ofCompound 6 (@3 mg/kg), and (c) DK-I-56-1 (@ 3 mg/kg).

Half animals (n=48) will be used for assessed by combined studies ofFos-immunoreactivities and tinnitus behavioral test and the rest animalwill be used for electrophysiological studies. Animals forelectrophysiological measurement will first undergo aseptic surgeryunder general anesthesia (isoflurane) before the sound or shamtreatment.

Loud sound exposure. The narrow band noise with the center frequency at10 kHz will be chosen since the 10 kHz is the most sensitive frequencyof the rat audiogram (Kelly and Mastron, 1977). Sound will be presentedto animals through a free-field speaker (TDT, FF1) placed at the ceilingof the sound-treated chamber (W×D×H=60×60cm×60cm³). An intensity levelof 120 dB SPL will be chosen and confirmed by the measurement with acalibration microphone (B&K 4149) placed at the position of animals. Thehead will be positioned approximately 40 cm below the center of thespeaker. The loud noise will be delivered to the animals for 2 hr. Ratsin group A will be anesthetized with 3% isoflurane during the soundexposure. Rats in group B will be anesthetized and put in the soundtreated room without sound exposure for 2 hrs.

After the acoustic manipulation, animals will be returned to normalrearing cages and environment until the subsequent experiment.

Surgery for chronic electrode implantation. For the implantation of thedorsal cochlear nucleus and auditory cortex (AC) recording electrode,twos hole (size ˜2 mm) will be made between bregma −10.80 to −11.76 and−3.3- to −6.3 mm for access to the DCN and AC respectively. The activerecording electrodes were made by Teflon-coated tungsten (A-M Systems,0.013″ OD) Teflon-coated silver (with tip made into a disk shape, A-MSystems, 0.013″ OD) wires, will be inserted into DCN and placed on thesurface AC. The reference electrodes will be implanted through anopening in the frontal skull. All wires will be connected to a miniatureconnector and fixed by Histoacryl and reinforced with dental cement (GCFuji Plus, Tokyo). The resected skin will be closed with fine suture(6/0). Wounds will be checked for possible infections on a daily basisand treated topically with antibiotic (chloramphenicol, 30 mg/kg, onceto twice daily when necessary).

Electrophysiological recordings. From 8 to 6 days before loud soundexposed or sham control treatment, all animals, with the recording wiresconnected will be preconditioned inside a behaving chamber (4 hrs/day)for preconditioning. Then, the control measurements of auditoryactivities will be made for 5 continuous days (4 hours/day: 1 hr insilence, 3 hrs for auditory threshold measurement). Both the spontaneousand sound evoked activities will be recorded from the AC. The effects ofloud sound exposure on AC activities will be will be measured for 7continuous days (4 hours/day: 1 hr in silence, 3 hrs for auditorythreshold measurement). Rats receiving (a) the vehicle, (b) Compound 6and (c) DK-I-56-1, measurements of spontaneous and sound evokedactivities will be again made in sessions each lasting for 6 hrs (2 hrsbefore to 4 hrs after the drug treatment).

During recording sessions, the miniature connector plug is connected toa chronic head stage (RA16CH, TDT), and fed to a 32-bit neurophysiologybase station (RZ5, TDT) that was controlled by an OpenEx software suite(TDT). Signals will be amplified, band-pass filtered (1-3000 Hz),monitored in real time and digitized at 12 kHz, 16-bit resolutions fordata storage. FIG. 2 shows the procedures of electrophysiologicalrecordings from the AC of behaving rats.

Acoustic stimuli. Tone bursts (25-msec duration, 2.5 msec rise/falltime) will be delivered through a free-field speaker (Fostex, FE103E)located at the ceiling of a sound-treated room where the animal ishoused. Tone bursts have a frequency chosen from a user menu (1, 2, 4,10, or 16 kHz). Sound intensity will be randomized for presentationwithin an intensity range (5 to 75 dB SPL, 5 dB steps). The intensitywill be calibrated at the site of animal from 0.5 to 30 kHz using aprecision microphone system (B&K 5113). Response to each tonepresentation will be collected on a single-trial basis for a duration of1 sec (400 msec before, 600 msec after stimulus onset). The inter trialinterval will be 2 sec. A total of 80-100 trials will be collectedduring a session at a fixed frequency and intensity. The silencesessions will be similarly conducted without the sound stimulation.

Data analysis. Single trials will be first processed by theevent-related analysis in the time and spectral domains. For time domainanalysis, the average evoked responses will be computed across repeatedtrials. The response-level function will be generated based on theevoked potential integrals obtained from two time windows (0-5 ms and15-200 msec).

Activates recorded from the AC will be also analyzed in spectral domain.The total power spectrum and the event related spectral perturbation(ERSP; Makeig, 1993) will be computed to reveal the spontaneousactivities and time-locked (but not necessary phase-lock) responses tosound. For the ERSP analysis, the baseline spectrum preceding thestimulus onset will be canceled from the post-stimulus response spectrumto suppress frequency components that are not related to the stimulussound. Specifically, short time fast Fourier transforms (FFT) will beapplied systematically with a running window (256 sample points, 244points in overlap). In the final step, non-significant parts of ERSP aretruncated to zero values through a procedure of bootstrapping. Thesingle trial will be first divided into 200 segments each of 512-pointlong. Each segment will be convolved with a Hanning window and then zeropadded to 1024 points for spectral estimation with FFT. Log powerspectra will be computed and normalized against the baseline (straighttunnel segment) log mean power spectra. The time series of each trialwill be entered into a time-frequency matrix (1000×2014), with afrequency resolution of 0.05 Hz. Results will be averaged acrossrepeated trials to yield a standard image of ERSP. Significant levels ofdeviations from pre-stimulus baseline will be assessed by bootstrapping(a nonparametric permutation-based statistical method). Non-significantpoints will be masked to zero to better reveal the perturbations withsignificance (p<0.05). The inter-trial coherence will be furtherdetermined to reflect neural synchrony. To characterize changes inspontaneous activities, the activities collected in the silence sessionswill be analyzed in the spectral domain (total spectrum) by FFT. Themagnitude of individual frequency components (0-3,000 Hz) will bepresented in dB scale. To reveal tinnitus-related changes in the restingstate, the power spectra of the SS treatment will be normalized againstthe control power spectra.

Statistical Analysis. Since the power spectra of silence and soundevoked sessions are not normally distributed, nonparametric statistictests are used for their analysis. Friedman's test (Matlab statisticaltoolbox, Mathworks) is used to evaluate the effects of tinnitus perceptor sound intensity on EPs. Bootstrapping (EEGLAB toolbox, UCSD) will beused for assessing statistical significance of power changes. To testgroup statistics, the intrinsic inter-animal EP difference will bereduced by dividing the grand mean of the silence trials (or averagedresponses) within the first 200 msec of each trial. The significantlevel is set at p<0.05.

Histological confirmation of cortical recording sites. On day followingthe end of recording, animals will be euthanized (Nembutal 75 mg/kg) andperfused with 0.9% saline solution followed by 4% paraformaldehyde(Merck), with the removed brains post-fixed overnight, cryoprotected in30% sucrose, 0.1 M phosphate buffered saline (PBS) solution. Brains willbe sectioned (40 μm) with a freezing microtome in the coronal plane, andplaced into 0.1 M PBS for later histology. Sections will be mounted onslides, air-dried, mounting with Vectashield medium (VectorLaboratories). To check the electrode positions, low magnifications(×40) images will be digitized (Canon E50) through a microscope (OlympusBX51). The location of electrode will be checked with the Di-I staintogether with the location of AC determined by landmarks referenced tothe rat brain atlas.

Behavioral test. The paradigm is modified from the Berger et al (2013).The background noise of gap detection will comprise with narrow-bandnoise (2 kHz bandwidth) with center frequencies varied at 4, 8 and 16kHz. Evoked stimuli will be short broad-band noise bursts (20 ms;rise/fall time of 1 ms). A single session will consist of 5presentations of the stimulus preceded by a silence gap, and 5presentations without a silence gap randomly delivered in a givenbackground noise condition. To avoid habituation, the inter stimulusinterval (ISI) will be around 10 min, leading to a single trial takingaround 1.5-2 hr. Gap duration of 100 ms (rise/fall time of 2 ms) and adelay of 100 ms between the gap onset and the startle stimulus onsetwill be used. The background noise will be randomly varied from 5 to 25dB SPL (5 dB/step) and the evoked stimulus will be presented at either10 dB SPL louder than the background. The pinna reflex will be measuredwith a high-resolution video camera system (Image Solution Group modelLW-1.3-S-1394-M) and data will be analyzed off-line using custom-madesoftware we developed on movement detection (details see Wu et al.,2000). The baseline of pinna reflex and Gap pre-pulse inhibition will bemeasured in each animal (at least 6 sessions over a week) before thesound exposure. Effects of mild sound exposure on pinna reflex and Gappre-pulse inhibition will be subsequently measured at 1-2 weeks afterthe termination of sound exposure. After finishing the behavioralassessments, animals will be sacrificed and their brains will beprocessed with Fos-immuno-histochemical studies.

Fos Immuno-histochemical staining. One day after finished the behavioralassessment, the experimental and control rats will be preconditioninginside the sound treated room for 8 hrs. Then, animals will besacrificed with overdose urethane (2.0 g/kg) and perfused via anintra-aortic catheter (with physiological saline PBS; pH 7.2, followedby 4% para-formaldehyde solution). The removed brains will becryoprotected overnight in a sucrose-buffer (30% in PBS). Frozensections (40 μm) will be cut in coronal plane and alternated sectionsincubated in solutions as listed below, interspersed PBS rinses: (a)normal goat serum at a dilution of 1:100 (Vector) for 2 hrs; (b) Fosantibody (1:4000, Santa Cruz) in PBS for 48 hrs at 4° C.; (b) PBS wash3×5 min; (c) biotinylated anti-rabbit IgG or biotinylated anti-mouse IgG(Vector) at 1:500 in PBS with 1% normal goat serum, incubated for 3 hrs;(d) rinsed with PBS 3×5 min; (e) incubated with ABC Kit reagent (Vector)for 2 hrs; (f) rinsed with PBS 3×5 min; (g) 3× rinses with acetatebuffer (0.1M, pH=6.0); (h) developed with nickel-DAB-glucose oxidasesolution for 5-10 min; (I) 2× rinses in acetate buffer for 5 min. Thesections will be mounted on the slide for the microscopic analysis.

All sections will be analyzed using light microscopy. Digitalphotomicrographs will be taken using a digital camera (Canon E50)mounted on an Olympus microscope (BX 51). Pictures will be digitallyadjusted for color, brightness, or contrast at the time that thephotograph was taken, but no further digital adjustments will be made tothe photograph of the tissue. High magnification (100×) composite imageswill be created by compiling photographic stacks using Image Pro Plussoftware (Media Cybernetics, Silver Spring, Md., USA). Locations oflabeled cells will be manually digitized (SummaSketch III, Xu et al.,1990) and reconstructed with software specially developed to visualizetheir 3D patterns of distribution (Wu et al., 2003). This approach willallow the examination of all labeled neurons along the auditory relaysfrom CN to AC.

The role of α6GABA_(A)Rs in depression. Finally, in the meantimeevidence accumulated indicating that α6-GABA_(A) receptors are widelydistributed throughout the brain, although with a much lowerconcentration than that found in the granule cells of cerebellum (Allenbrain atlas). It can be assumed that low abundance receptors exhibitquite specific and important functions in the brain by modulating onlythose neurons on which they are located.

Since linkage studies indicate that the gene for α6-subunits isassociated with female patients with mood disorders (Yamada et al.,2003), α6-containing receptors might also have some beneficial effectsin mood disorders such as depression. Experiments are underwayinvestigating whether compounds in this patent application are able toameliorate symptoms of depression in several animal models ofdepression.

1. A compound according to Formula I:

wherein each X is independently C or N; R′₂, R′₃ and R′₄ areindependently selected from H, C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen,NR₁₀R₁₁, NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or—C(O)NR₁₀R₁₁; R₆, R₇, R₈ and R₉ are independently selected from H, C₁₋₄alkyl, C₁₋₄ alkoxy, halogen, NR₁₀R₁₁, NO₂, hydroxyl, cyano, C₁₋₄alkylthio, —C(O)C₁₋₄ alkyl, or —C(O)NR₁₀R₁₁, or R₆ and R₇ or R₇ and R₈can form a 4-6 member ring; R₁₀ and R₁₁ are independently selected fromH and C₁₋₄ alkyl; and R_(N) is H or C₁₋₄ alkyl; with the provisio thatthe compound is not


2. A compound according to Formula (II):

wherein each X is independently C or N; R′₂, R′₃ and R′₄ areindependently selected from H, C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen,NR₁₀R₁₁, NO₂, hydroxyl, cyano, C₁₋₄ alkylthio, —C(O)C₁₋₄ alkyl, or—C(O)NR₁₀R₁₁; R₆, R₇, R₈ and R₉ are independently selected from H, C₁₋₄alkyl, C₁₋₄ alkoxy, and halogen; NR₁₀R₁₁, NO₂, hydroxyl, cyano, C₁₋₄alkylthio, —C(O)C₁₋₄ alkyl, or —C(O)NR₁₀R₁₁; or R₆ and R₇ or R₇ and R₈can form a 4-6 member ring; R₁₀ and R₁₁ are independently selected fromH and C₁₋₄ alkyl; and R_(N) is H or C₁₋₄ alkyl; wherein at least one ofR′₂, R′₃, R′₄, R₆, R₇, R₈ , R₉, R₁₀, R₁₁ and R_(N) contains at least onedeuterium.
 3. A compound according to claim 1 or claim 2, wherein atleast one of R′₂, R′₃, R′₄, R₆, R₇, R₈ R₉, R₁₀, R₁₁ and R_(N) is ahaloalkyl.
 4. The compound according to claim 3, wherein the haloalkylis CF₃.
 5. A compound according to any of the preceding claims whereinat least one of R′₂, R′₃, R′₄, R₆, R₇, R₈ , R₉, R₁₀, R₁₁ and R_(N) isC₁₋₄ alkoxy.
 6. A compound according to claim 5, wherein the C₁₋₄ alkoxyis a methoxy.
 7. A compound according to claim 6, wherein the C₁₋₄alkoxy is —OCD₃.
 8. A compound according to any of the preceding claimswherein at least one of R′₂, R′₃, R′₄, R₆, R₇, R₈, and R₉ is a halogen.9. A compound according to claim 8 wherein the halogen is bromine orchlorine.
 10. A compound according to any of the preceding claimswherein each X is C.
 11. A compound according to any of claims 1-9wherein at least one X is N.
 12. A compound according to any of thepreceding claims wherein R′₄ is selected from H, —OCH₃ and —OCD₃.
 13. Acompound according to any of the preceding claims wherein R′₃ isselected from H, —OCH₃ and —OCD₃.
 14. A compound according to any of thepreceding claims wherein R₆ is selected from H, —OCH₃ and —OCD₃.
 15. Acompound according to any of the preceding claims wherein R₇ is selectedfrom H, halogen, —OCH₃ and —OCD₃.
 16. A compound according to any of thepreceding claims wherein R₈ is selected from H or halogen.
 17. Acompound according to any of the preceding claims wherein R_(N) is H.18. A compound according to any of the preceding claims selected fromthe group consisting of:


19. A compound according to any of the preceding claims selected fromthe group consisting of:


20. A compound according to any of the preceding claims selected fromthe group consisting of:


21. A compound according to any of the preceding claims selected fromthe group consisting of: wherein R₁ and R₂ are independently H, OCH₃,OCD₃, OEt, OCF₃, F, Cl, BR, or NO₂.
 22. A compound according to any ofthe preceding claims selected from the group consisting of:


23. A pharmaceutical composition comprising a compound according to anyof the preceding claims and a pharmaceutically acceptable carrier.
 24. Apharmaceutical composition comprising a compound and a pharmaceuticallyacceptable carrier; wherein the compound is


25. A method of treating diseases and/or conditions which are regulatedby the α6-GABA_(A) receptor comprising administering a therapeuticallyeffective amount of a compound according to any one of claims 1-22 or apharmaceutical composition according to claim 23 to a subject in needthereof.
 26. A method of treating a disease and/or condition comprisingadministering a therapeutically effective amount of a compound accordingto any one of claims 1-22 or a pharmaceutical composition according toclaim 23 to a subject in need thereof; wherein the disease is modulatedby the α6-subunit GABA_(A) receptor.
 27. A method of treating diseasesand/or conditions which are regulated by the α6-GABA_(A) receptorcomprising administering a therapeutically effective amount of acompound selected from

or a pharmaceutical composition according to claim 24 to a subject inneed thereof.
 28. A method of treating a disease and/or conditioncomprising administering a therapeutically effective amount of acompound selected from

or a pharmaceutical composition according to claim 24 to a subject inneed thereof; wherein the disease is modulated by the α6-subunitGABA_(A) receptor.
 29. The method of claim 26 or 28, wherein the diseaseor condition is selected from the group consisting of neuropsychiatricdisorders with sensorimotor gating deficits; depression;temporomandibular myofascial pain; disorders of trigeminal nerve; ;migraine; and tinnitus.
 30. The method of claim 29, wherein theneuropsychiatric disorder is selected from the group consisting ofschizophrenia, tic disorders, attention deficit hyperactivity disorder,obsessive compulsive disorder, panic disorder, Huntington's disease andnocturnal enuresis.
 31. The method of claim 29, wherein the disorder oftrigeminal nerve is selected from the group consisting of trigeminalneuralgia and trigeminal neuropathy.